MÁQUINAS DE APRENDIZAJE EXTREMO MULTICAPA (ML-ELM)

publications, BMMφ were cultured under the same conditions and analysed for the mRNA levels of type I (M1) (NOS2) or type 2 (M2)- (Arg-1, Chi3l3and MRC1) associated mediators following priming by the M1-inducing cytokine, IFN-Υ, or M2-inducing cytokine, IL-4 respectively. The increased levels of these mediators in response to their corresponding inducing cytokines would serve as a positive control for further cytokine-priming experiments in AMφ polarisation, which is the primary objective of these experiments. However, it should be noted that the mRNA levels of M2-associated mediators were not evaluated in M1, nor M1-associated mediators in M2 BMMφ. In other words, these experiments only reveal whether or not priming signals can induce a change in mediator mRNA levels in BMMφ, but cannot fully characterise BMMφ polarisation (whether or not M1 or M2 BMMφ have been generated).

Chapter 3| The heterogeneity of AMφ in vitro

In the BMMφ-priming experiments, IFN-Υ-primed BMMφ displayed increased NOS2 mRNA levels, whereas Arg-1, Chi3l3, and MRC1 mRNA levels was elevated in IL-4 primed BMMφ (Figure 3.1).

These results demonstrate that as reported in the literature282,340,738,739, BMMφ increase different gene transcripts in response to different cytokine priming.

Figure 3.1 Effect of cytokine priming on mRNA levels of macrophage subset markers in Bone Marrow Derived Macrophages (BMMφ).1 x 10⁵ BMMΦ were seeded per well of a 48-well culture plates, and primed with macrophage complete media control (RPMI-1640 + 10% fetal bovine serum + 1.5 mM sodium pyruvate + 0.05 mM β-mercaptoethanol + 1 mM penicillin/streptomycin + 50 μg/ml geneticin) alone or together with 20 ng/ml Interferon-Υ (IFN-Υ) or 10 ng/ml Interluekin-4 (IL-4) for 22 hours. Nitric Oxide Synthase (NOS) 2, Arginase-1 (Arg-1), Mannose Receptor C, type 1 (MRC1), and Chitinase 3 like 3 (Chi3l3) mRNA levels were measured by Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Data are from one pilot study collected from pooled samples run in triplicates and the average was presented as relative % to the housekeeping gene Hypoxanthine Phosphoribosyltransferase 1 (HPRT-1). ND: Not detected.

3

3.5.1.2.2 Differentially primed BMMφ have distinct responses to LPS stimulation

TLR activation is generally ascribed to modifying macrophages to promote a pro-inflammatory phenotype. However this phenotype may be differentially regulated in various macrophage subsets.

M1 macrophages have been described to promote inflammation through the release of pro-inflammatory mediators, ROS, and RNS, and augment the pro-pro-inflammatory response following TLR stimulation. M2 macrophages have a wound healing phenotype that is characterised by Arg-1 expression^581,582 and expression of various wound healing mediators (e.g. PDGF and

fibronectin)^120,520, but have also been reported to have increased expression of pro-inflammatory mediators in response to LPS stimulation⁷³⁸.

In order to validate these observations, a pilot study was carried out. M1 and M2 macrophages were generated as before by priming BMMφ with IFN-Υ or IL-4; these were then further stimulated with LPS for TLR4 activation.

Chapter 3| The heterogeneity of AMφ in vitro

3.5.1.2.2.1 IFN-Υ primed macrophages have an exaggerated pro-inflammatory response to LPS stimulation

As has been reported by others282,340,738,739, LPS stimulation promoted a pro-inflammatory response and increased both NOS2 and IL-1β gene transcript levels. IFN-Υ-primed macrophages have an exaggerated response to LPS stimulation, which strongly augmented increased transcript levels of NOS2 by around 4-fold and to a lesser extent IL-1β. On the other hand, IL-4-primed macrophages have a dampened pro-inflammatory response to LPS stimulation and reduced LPS-induced increase in IL-1β mRNA levels (Figure 3.2).

As has been reported by others283,341,738,739, LPS stimulation promoted a pro-inflammatory response and increased both NOS2 and IL-1β gene transcript levels. IFN-Υ-primed macrophages have an exaggerated response to LPS stimulation, which strongly augmented increased transcript levels of NOS2 by around 4-fold and to a lesser extent IL-1β. On the other hand, IL-4-primed macrophages have a dampened pro-inflammatory response to LPS stimulation and reduced IL-1β upregulation (Figure 3.2).

Figure 3.2 The effects of Lipopolysaccharide (LPS) stimulation on mRNA levels of M1-associated markers in differentially primed BMMφ.1 x 10⁵ BMMΦ were seeded per well of a 48-well culture plates, and primed with macrophage complete media control (RPMI-1640 + 10% fetal bovine serum + 1.5 mM sodium pyruvate + 0.05 mM β-mercaptoethanol + 1 mM penicillin/streptomycin + 50 μg/ml geneticin) alone or together with 20 ng/ml Interferon-Υ (IFN-Υ) or 10 ng/ml Interluekin-4 (IL-4) for 18 hours. Primed BMMΦ were then further cultured with macrophage complete media control or 10 ng/ml LPS from E. coli 0111:B4 for 4 hours. NOS2 and IL-1β mRNA levels were measured by qRT-PCR. Data are from one pilot study collected from pooled samples run in triplicates and the average was presented as relative % to the housekeeping gene HPRT-1.

ND: Not detected.

Co n

3.5.1.2.2.2 LPS stimulation shifts macrophage subset bias away from M2 phenotype

LPS stimulation promoted a pro-inflammatory phenotype, and was synergistic to increased levels of NOS2 and IL-1β gene transcripts in BMMφ induced by IFN-Υ priming (Figure 3.2). In contrast, LPS stimulation shifts the macrophage subset bias away from an IL-4 primed phenotype, as observed by the decline in Arg-1 and Chi3l3 transcript levels in IL-4-induced BMMφ by approximately 25% and 50% respectively (Figure 3.3). These results demonstrate that LPS stimulation of TLR4 may alter macrophage phenotype driven by cytokine priming.

Figure 3.3 The effects of Lipopolysaccharide (LPS) stimulation on mRNA levels of M2-associated markers in differentially primed BMMφ.1 x 10⁵ BMMΦ were seeded per well of a 48-well culture plates, and primed with macrophage complete media control (RPMI-1640 + 10% fetal bovine serum + 1.5 mM sodium pyruvate + 0.05 mM β-mercaptoethanol + 1 mM penicillin/streptomycin + 50 μg/ml geneticin) alone or together with 20 ng/ml IFN-Υ or 10 ng/ml IL-4 for 18 hours. Primed BMMΦ were then further cultured with macrophage complete media control or 10 ng/ml LPS from E. coli 0111:B4 for 4 hours. Arg-1 and Chi3l3 mRNA levels were measured by qRT-PCR. Data are from one pilot study collected from pooled samples run in triplicates and the average was presented as relative % to the housekeeping gene HPRT-1.

Chapter 3| The heterogeneity of AMφ in vitro

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