Comparing Effects of Transforming Growth Factor β1 on Microglia From Rat and Mouse: Transcriptional Profiles and Potassium Channels

Abstract

The cytokine, transforming growth factor β1 (TGFβ1), is up-regulated after central nervous system (CNS) injuries or diseases involving microglial activation, and it has been proposed as a therapeutic agent for treating neuroinflammation. Microglia can produce and respond to TGFβ1. While rats and mice are commonly used for studying neuroinflammation, very few reports directly compare them. Such studies are important for improving pre-clinical studies and furthering translational progress in developing therapeutic interventions. After intracerebral hemorrhage (ICH) in the rat striatum, the TGFβ1 receptor was highly expressed on microglia/macrophages within the hematoma. We recently found species similarities and differences in response to either a pro-inflammatory (interferon-γ, IFN-γ, +tumor necrosis factor, TNF-α) or anti-inflammatory interleukin-4 (IL-4) stimulus. Here, we assessed whether rat and mouse microglia differ in their responses to TGFβ1. Microglia were isolated from Sprague-Dawley rats and C57BL/6 mice and treated with TGFβ1. We quantified changes in expression of >50 genes, in their morphology, proliferation, apoptosis and in three potassium channels that are considered therapeutic targets. Many inflammatory mediators, immune receptors and modulators showed species similarities, but notable differences included that, for some genes, only one species responded (e.g., Il4r, Il10, Tgfbr2, colony-stimulating factor receptor (Csf1r), Itgam, suppressor of cytokine signaling 1 (Socs1), toll-like receptors 4 (Tlr4), P2rx7, P2ry12), and opposite responses were seen for others (Tgfb1, Myc, Ifngr1). In rat only, TGFβ1 affected microglial morphology and proliferation, but there was no apoptosis in either species. In both species, TGFβ1 dramatically increased Kv1.3 channel expression and current (no effects on Kir2.1). KCa3.1 showed opposite species responses: the current was low in unstimulated rat microglia and greatly increased by TGFβ1 but higher in control mouse cells and decreased by TGFβ1. Finally, we compared TGFβ1 and IL10 (often considered similar anti-inflammatory stimuli) and found many different responses in both species. Overall, the numerous species differences should be considered when characterizing neuroinflammation and microglial activation in vitro and in vivo, and when targeting potassium channels.

Microglia Responses to Pro-inflammatory Stimuli (LPS, IFNγ+TNFα) and Reprogramming by Resolving Cytokines (IL-4, IL-10)

“…We find that these microglia have very low expression of many inflammatory mediators that are characteristic of more activated cells (e.g., Sivagnanam et al, 2010 ; Liu et al, 2013 ; Lam and Schlichter, 2015 ; Siddiqui et al, 2016 ; Lively et al, 2018 ). Of course, they are not “quiescent.” For instance, as appropriate for neonatal microglia that are involved in refining the brain architecture, many are unipolar with a large lamellum and a uropod and are highly migratory ( Lively and Schlichter, 2012 , 2013 ; Siddiqui et al, 2012 , 2016 ; Vincent et al, 2012 ; Lam et al, 2017 ; Lively et al, 2018 ). The cerebellum was removed and the remaining brain tissue was minced in cold Minimal Essential Medium (MEM; ThermoFisher Scientific, RRID :SCR_008452; Cat# 11095080), strained and centrifuged at 300 × g for 10 min.…”

“…Microglia were isolated from 1 to 2-day old Sprague-Dawley rat pups (Charles River, St.-Constant, PQ, Canada) using standard operating protocols that we find yield essentially pure microglia, as determined by labeling with tomato lectin, isolectin B4, or antibodies against Iba1 or CD11b ( Cayabyab et al, 2000 ; Khanna et al, 2001 ; Ducharme et al, 2007 ; Ohana et al, 2009 ; Schlichter et al, 2010 ; Sivagnanam et al, 2010 ; Lively and Schlichter, 2013 ; Lam and Schlichter, 2015 ; Siddiqui et al, 2016 ; Lively et al, 2018 ). Anti-CD11b staining of the present cultures is shown in Supplementary Figure 1A .…”

“…Western blotting was used to determine whether observed mRNA changes correlated in time with protein changes, as before ( Lam et al, 2017 ; Lively et al, 2018 ). Microglia were seeded on 25 mm coverslips (Fisher Scientific, Ottawa, ON, Canada; Cat# 12-545-86) in 35 mm culture dishes at 1–3 × 10 6 cells (5–6 independent cell cultures).…”

“…To compare changes in protein levels, total protein normalization was used, as before ( Lam et al, 2017 ; Lively et al, 2018 ). Membranes were stained for 1 min with 0.1% Coomassie Brilliant Blue G (Sigma-Aldrich; Cat# B8522), de-stained for 2 min in acetic acid/methanol/water (1:5:4), air-dried, and imaged with a ChemiDocTM XRS System.…”

Involvement of Wnt7a in the role of M2c microglia in neural stem cell oligodendrogenesis

“…For in vitro studies, microglial cells were seeded at a density of 100,000 cells/cm 2 and they were lysed 6 h or 24 h after stimulation with known agents that induce the polarization of microglia to obtain M1 (LPS), M2a (IL-4 and IL-13), and M2c (TGFβ) microglia [35,36]. In another subset of experiments, the microglia cells were lysed 6 h after the addition of purified myelin (2 μg/ml).…”