Inflammation ( # 2015) DOI: 10.1007/s10753-015-0136-y

Macrophage-Microglia Networks Drive M1 Microglia Polarization After Mycobacterium Infection Yongwei Qin,1 Xiaolei Sun,1 Xiaoyi Shao,1 Chun Cheng,1 Jinrong Feng,1 Wei Sun,1 Delin Gu,2 Wei Liu,1 Feifan Xu,2 and Yinong Duan1,3

Abstract—Central nervous system tuberculosis (CNS-TB) is caused by infection with Mycobacterium tuberculosis (Mtb). The inflammatory response following CNS-TB involves the activation of resident microglia and the infiltration of macrophages. However, it has not been clarified whether microglia can be polarized into the classically activated proinflammatory M1 phenotype or the alternatively activated anti-inflammatory M2 phenotype after Mtb infection. In this study, we found that BV2 treated with conditioned media from cultures of macrophages infected with Mycobacterium marinum (Mm) induced the expression of M1 phenotypic genes including iNOS, TNF-α, IL-1β, IL-6, CCL2, and CXCL10 but reduced that of M2 phenotypic genes such as Arginase 1, Ym1, and CD163. These results suggest that polarization of microglia is partly mediated through macrophage-microglia interactions as a priming signal. Overall, these results provide new insights into the modulatory mechanisms of microglial polarization, thereby possibly facilitating the development of new therapies for CNS-TB infection via the regulation of microglial polarization through signalling derived from macrophages infected with mycobacteria. KEY WORDS: classically activated; microglia; Mycobacteria CNS-TB.

INTRODUCTION Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains one of the major public health problems. One third of the world’s population is estimated to be latently infected with Mtb, with 8.7 million cases of diagnosed disease and 1.4 million deaths occurring each year (WHO Tuberculosis Data and Statistics, 2011). Central nervous system tuberculosis (CNS-TB), mostly tuberculous meningitis (TBM), is the most severe extra pulmonary complication of TB and accounts for approximately 5–15 % of the extra pulmonary cases. In developing countries, CNS-TB occurs primarily in childhood Yongwei Qin and Xiaolei Sun contributed equally to this work. 1

Department of Pathogen and Immunology, Medical College, Nantong University, 19 Qixiu Road, Nantong, 226001Jiangsu, People’s Republic of China 2 The Sixth People’s Hospital of Nantong, 500 Yonghe Road, Nantong, 226011, China 3 To whom correspondence should be addressed at Department of Pathogen and Immunology, Medical College, Nantong University, 19 Qixiu Road, Nantong, 226001Jiangsu, People’s Republic of China. E-mail: [email protected]

but significantly increases in adults under conditions of immune suppression, which are associated with considerable morbidity and mortality [1, 2]. Pathogenesis of CNS-TB is initiated as a secondary infection during hematogenous dissemination of pulmonary infection to the brain parenchyma [3]. Despite the neuroprotective properties of the blood brain barrier (BBB), it has been proposed that Mtb are able to cross this BBB with relatively high efficiency and invade the CNS as free bacilli [4, 5]. In recent years, the concept of macrophage polarization has been developed and further sophisticated: macrophages are now seen as a spectrum of phenotypic subtypes ranging from classically activated macrophages (M1) to the alternatively activated macrophages (M2) based on gene expression induced in response to pathogen and cytokine stimulation [6, 7]. The functions of M1 are geared towards the killing of intracellular pathogens and antigen presentation. For example, MHC II, Fcγ receptors, and CD86 are upregulated to allow for antigen-presenting activity of microglia and increased crosstalk with other immune cells [8]. Another potential distinction and an important component of M1 microglia is their ability to produce reactive oxygen species and reactive nitrogen species [9].

0360-3997/15/0000-0001/0 # 2015 Springer Science+Business Media New York

Qin, Sun, Shao, Cheng, Feng, Sun, Gu, Liu, Xu, and Duan M2 macrophages play a critical role in the resolution of inflammation and formation of granuloma by producing anti-inflammatory mediators and collagen. The resident CNS macrophages, microglias, are the principal target cells in CNS-TB, and they are efficiently infected with Mtb [10]. Activated microglias secrete many chemokines and cytokines that exert neuroprotective or neurotoxic effects [11]. These cytokine and chemokine production are essential for the polarization of microglia into what has been termed a classical activation or alternative activation state [12]. M1 phenotype includes production of nitric oxide (NO), TNFα, and IL-1, 6, 12. The alternative activation, M2 state, phenotype is characterized by expression of Arginase 1 (Arg 1), chitinase 3-like 3 (Ym1), found in inflammatory zone 1 (FIZZ1), CD163, dectin-1, mannose receptor, and IL-10 [13]. Microglia can adopt a distinct phenotype in response to Th1 or Th2 cytokines (IFN-γ/TNFα vs. IL-4/IL-13) [14]. Microglias are able to control their own polarization through autocrine and paracrine means. In many cases, this response is protective and is downregulated once the inflammatory damage or pathogen has been dealt with; however, unregulated, long-term, or chronic inflammation can lead to tissue destruction [15]. In the early stage infection in CNS-TB, little is known about the polarize state of miroglia driven by the signals derived from macrophages infected with mycobacteria. Mycobacterium marinum (Mm) is one of the closest relatives of members of the Mtb that can eventually infect humans. Similarly to Mtb, after infecting host macrophages, Mm causes a tuberculosis-like disease in fish and amphibians. Human infections are limited to cool surfaces, such as skin and extremities [16]. It has an in vitro growth rate that is three to five times faster than that of Mtb. Mm can be easily manipulated in a Biosafety Level (BSL)-2 laboratory. Furthermore, Mm and Mtb share a lot of important virulence factors, of which the ESX-1 locus is one of the best examples. All these features have made Mm a widely accepted model to study mycobacterium infections [17]. As such, Mm infection model is a reproducible and accessible model to analyze the pathogenesis of early CNS-TB granuloma formation [5]. In this study, we investigated the potential of macrophage-microglia network drive M1 or M2 microglia polarization after mycobacterium infection. We show that microglia was modulated toward M1 polarization by the priming signals derived from macrophages infected with mycobacteria.

METHODS Cell Culture Mouse primary microglial cultures were performed as described previously [18]. Briefly, the neonatal mouse cerebral cortex was dissociated and plated in tissueculture flasks. Ten days later, the confluent glial mixed cultures were treated with mild trypsinization (0.05 % trypsin with 0.25 mM EDTA) for 5 min that detaches a layer of loosely adherent astrocytes and leaves behind firmly adherent cells, which are >90 % microglia. Afterward, cells were cultured for 5 days in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, Grand Island, NY, USA) culture medium containing 10 % fetal bovine serum (FBS) (Gibco), 1 % penicillin/streptomycin (Gibco), and 1 % vitamins (Gibco) mixed 1:1 with mixed glial cellconditioned media before stimulation. The immortalized murine microglia cell line BV-2 and the mouse macrophage cell line RAW 264.7 cells (Cell Resource Center of Peking Union Medical College, Beijing, China) were, respectively, maintained in DMEM/F12 or DMEM (Gibco) supplemented with 10 % FBS (Gibco), 1 % penicillin/streptomycin (Gibco), at 37 °C in a humidified atmosphere of 95 % air and 5 % CO2. Bacteria Mycobacterium marinum strain M (ATCC, Manassas, VA, USA) were grown in Middlebrook 7H9 broth (Difco, Franklin Lakes, NJ, USA) supplemented with 10 % Middlebrook oleic acid-albumin-dextrose-catalase (OADC) (BD Biosciences, NZ, USA) and 0.05 % Tween 80, and 0.5 % glycerol at 32 °C. For infection experiments, bacteria were grown to logarithmic phase, washed, and diluted in DMEM (Gibco). To remove bacterial clumps, bacterial suspensions were subjected to low-speed centrifugation (300×g, 10 min) after which the supernatant was passed through a 5-μm sterile filter (Millipore, Molsheim, France) and used at an indicated multiplicity of infection (MOI). Preparation of CoMm and CoU RAW 264.7 cells were infected with Mm at a MOI of 10 or were used uninfected [19]. After 24 h of incubation, cell culture supernatants were harvested and filtered through a 0.2-μm sterile filter (Millipore) [20]. The conditioned medium from Mm-infected RAW 264.7 cells were termed CoMm, and conditioned medium from Mm-uninfected RAW 264.7 cells were termed CoU. Untreated cells were termed U. The CoU and CoMm (1:10 dilution) were

Microglial M1 polarization in CNS-TB used to prime microglia for 4 h, washed, and then cultured with 10 % FBS DMEM/F12 (Gibco) medium for indicated times in all of the experiments. In some experiments, microglia were treated with lipopolysaccharide (LPS) (100 ng/mL) (Sigma-Aldrich, St Louis, MO, USA) plus IFN-γ (50 U/mL) (Peprotech, Rock Hill, NJ) or IL-4 (10 ng/mL) (Peprotech) for indicated times. RNA Isolation and Real-Time PCR Quantifications Total RNA was isolated from BV2 using RNeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. First-strand cDNAs were synthesized using a Thermo-script RT kit (Life Technologies, Rockville, MD, USA). PCRs were carried out in a Bio-Rad DNA Engine. Quantitative real-time PCRs (qPCRs) were performed in a CFX96TM Real Time System (Bio-Rad, Hercules, CA, USA), using SYBR green PCR reagent kits (Takara, Shiga, Japan) for mRNA transcript quantitation. Relative amount of transcripts was calculated using the 2−ΔΔCt formula. All PCR primers are reported in Table 1.

Minneapolis, MN, USA), and IL-6 (BioLegend, San Diego, CA, USA) using ELISA according to the manufacturer’s instructions. Griess Test Culture supernatant 50 μl and standards was transferred to a flat bottom 96-well plate, and then 50 μl Griess reagent was added. After incubation for 5 min in the dark at room temperature, the plate was read at 540 nm and nitrate concentrations were calculated. Statistical Analysis Data are expressed as means ± SEM of the indicated number of independent experiments. Statistical significance between multiple groups was analyzed by one-way ANOVA. The P values

Macrophage-Microglia Networks Drive M1 Microglia Polarization After Mycobacterium Infection.

Central nervous system tuberculosis (CNS-TB) is caused by infection with Mycobacterium tuberculosis (Mtb). The inflammatory response following CNS-TB ...
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