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Almost all cell types can express TLRs, but their response to TLR activation may vary. As reviewed in Section 4.1, different adipose tissue components can express TLR9. Thus some large adipocytes can display a pro-inflammatory phenotype, but the role of TLRs in this phenotypic switch has not been fully characterized. In the present study, transcript expression of TLR9 and some other TLRs and signalling molecules were measured in visceral adipose tissue and its cellular components.

As described in Section 4.1, TLR9 recognizes oligonucleotides (ssDNA fragments) from necrotic cellular debris, whereas TLR3 recognizes dsRNA and TLR4 recognizes LPS. There were increased amounts of Tlr9 mRNA in the visceral WAT of atherogenic diet-fed foz/foz mice vs. WT (Fig. 4.12A). As expected (by definition), Tlr9 mRNA expression was virtually absent in visceral WAT of atherogenic diet-fed Tlr9-/- mice. On the other hand, Tlr3 mRNA expression was higher in visceral WAT of atherogenic diet-

155 fed Tlr9-/- compared to WT mice (Fig. 4.12B). There were no significant changes in Tlr4 mRNA between any of the groups (Fig. 4.12C). There was a possible increase in Trif mRNA in atherogenic diet-fed foz/foz mice visceral WAT (not significant) (Fig. 4.12D), but MyD88 mRNA levels were clearly similar in all genotypes (Fig. 4.12E).

Figure 4.12: Visceral WAT mRNA expression of Tlr9, Tlr3, and Tlr4 as well as their

adapter proteins, Trif and MyD88, in atherogenic diet-fed mice. Data are mean ± SEM

(n=8/gp). Values are expressed relative to WT, which is set at 1.0.

† P<0.05 vs. WT control (genotype effect); e.g., atherogenic diet-fed foz/foz vs. WT

The increase in Tlr9 mRNA expression would be consistent with increased TLR9 protein levels in inflamed adipose tissue. To establish whether this is the case and to identify the cell type(s) responsible, we performed flow cytometry of mature adipocytes (Nile Red dye) and macrophages (fluorescence-labelled F4/80 antibody). As described in Section 2.3.3, cell solutions were prepared from a cohort of atherogenic diet-fed foz/foz mice (identical to the cohort of foz/foz mice studies for other read-outs). These preliminary data show that the number of TLR9-expressing adipocytes was low (~5%) in these three independent adipocyte samples (Fig. 4.13).

156

Figure 4.13: TLR9 expression in mature adipocytes from visceral WAT of atherogenic

diet-fed foz/foz mice. Flow cytometry analysis showed that lipid-filled, mature adipocytes from

visceral WAT (stained with Nile red) showed little, if any, bona fide expression of TLR9

protein. (SSC [side-scattered light] for single cell stream)

The same adipose tissues and a fourth sample were analysed for macrophage expression of TLR9 using a similar flow cytometry protocol. In this experiment, SVF cells were separated from adipocytes by the method outlined in Section 2.3.3. As mentioned in Section 1.5.6, pro-inflammatory (M1) macrophages express F4/80 protein on their extracellular surface. We therefore consider F4/80 to be a relevant marker to

157 identify macrophages in inflamed adipose tissue. Preliminary analyses of visceral WAT SVFs showed high levels of TLR9 expression on F4/80 positive cells. Accordingly, ~21 - 38% of SVF cells displayed both F4/80 and TLR9 expression (Fig. 4.14). In all, TLR9 expression levels were 4-8-fold greater in F4/80 positive macrophages than in Nile Red- stained adipocytes from visceral WAT of atherogenic diet-fed foz/foz mice.

Figure 4.14: F4/80 and TLR9 co-expression in stromal vascular cells from visceral WAT

of atherogenic diet-fed foz/foz mice. Visceral WAT stromal vascular cells expressing pro-

inflammatory marker, F4/80, also often expressed TLR9 protein (~21 – 38% of all cells).

One of main hypotheses of this PhD study is that degenerating adipocytes (which are small) attract inflammation into adipose tissue. As mentioned in Section 1.5, 90% of pro-inflammatory macrophages localize around such small adipocytes (Fig. 4.15A).

158 Accordingly, we measured the number of visceral WAT CLSs. The number of CLSs in visceral WAT increased slightly with atherogenic dietary feeding in WT mice (Fig. 4.15B,C), but Tlr9-/- mice were protected from the formation of CLS, even during intake of an atherogenic diet. In foz/foz mice, CLS recruitment already seemed evident on chow diet (not significant vs. WT), and the number of sites of inflammation (such as shown in Fig. 4.13A) was significantly higher in atherogenic diet-fed foz/foz mice visceral WAT compared to diet- or genotype-matched counterparts (Fig. 4.15B,C).

159

Figure 4.15: Number of CLSs in visceral WAT from WT, foz/foz and Tlr9-/- _{mice fed NC or}

Ath. (A) Numerous mononuclear inflammatory cells, abutting degenerating adipocytes to form

CLSs, are illustrated. (B,C) Such CLSs were abundant in foz/foz mice, especially with

atherogenic diet (n=8/gp [10 sections per mouse]; H&E-staining, 160x magnification). The

number of CLSs was small in atherogenic diet-fed WT mice, and they were virtually absent in

atherogenic diet-fed Tlr9-/- _{visceral WAT.}

Data are mean ± SEM (n=8/gp).

† P<0.05 vs. diet-matched WT (genotype effect); e.g., chow-fed foz/foz vs. WT

‡ P<0.05 vs. genotype-matched control (diet effect); e.g., atherogenic diet-fed vs. chow-fed WT

As stated in Section 4.1, IFNγ and RANTES contribute to adipose inflammatory recruitment. In the present study, Ifnγ and Rantes mRNA expression were both high in visceral WAT of atherogenic diet-fed foz/foz (not significant for Ifnγ) compared to WT mice (Fig. 4.16A,B), with no changes in Tlr9-/- mice. Ip10 mRNA expression appeared to be higher (not significant) in atherogenic diet-fed foz/foz mouse visceral WAT

160 compared to WT (Fig. 4.16C), with no difference for WT and Tlr9-/- mice. Compared to WT, there was no significant increase of Mcp1 mRNA in visceral WAT of atherogenic diet-fed foz/foz mice, but Tlr9-/- mice exhibited less Mcp1 mRNA than WT (Fig. 4.16D). There were no genotype differences in visceral WAT Chemerin mRNA expression (Fig. 4.16E).

Figure 4.16: Visceral WAT mRNA expression levels for genes related to macrophage

chemotaxis in atherogenic diet-fed mice. Data are mean ± SEM (n=8/gp). Values are

expressed relative to WT, which is set at 1.0.

† P<0.05 vs. WT control (genotype effect); e.g., atherogenic diet-fed foz/foz vs. WT

As mentioned in Section 1.5.6, CD11b is an important pro-inflammatory (M1) macrophage marker (318). In the present study, Cd11b mRNA levels were significantly higher in atherogenic diet-fed foz/foz visceral WAT than WT mice (Fig. 4.17A). Tlr9 deletion did not alter Cd11b mRNA expression levels. Cd11c, a marker of both macrophages and DCs (319), was increased in foz/foz mouse visceral WAT (Fig. 4.17B). Consistent with the vanishingly small number of recruited CLSs, Cd11c mRNA levels were very low in Tlr9-/- mice visceral WAT compared to WT. There seemed to be an increase in Icam mRNA in atherogenic diet-fed foz/foz mice visceral WAT, but this

161 was not significant compared to WT mice (Fig. 4.17C). In contrast, Tlr9-/- mice had significantly less Icam mRNA in visceral WAT in comparison to WT.

Figure 4.17: Visceral WAT mRNA expression levels for genes related to pro-inflammatory

macrophage infiltration into adipose in atherogenic diet-fed mice. Data are mean ± SEM

(n=8/gp). Values are expressed relative to WT, which is set at 1.0.

† P<0.05 vs. WT control (genotype effect); e.g., atherogenic diet-fed foz/foz vs. WT

4.4.5 Are Bone Marrow-Derived Cells Bearing TLR9 Essential for Adipose