Toll-like receptors and inflammation in metabolic neuropathy; a role in early versus late disease?

Abstract

Neuropathy is a common, morbid complication of the metabolic syndrome, prediabetes, and diabetes. Recent studies have indicated a potential role for the immune system in the development of neuropathy. In particular, toll-like receptors (TLR) 2 and 4 have been linked to metabolic dysfunction, and blocking TLR4 is proposed as a treatment for neuropathic pain. In the current study, we investigated the role of the immune system, particularly TLRs 2 and 4, in the pathogenesis and progression of neuropathy. Sural or sciatic nerve gene expression arrays from humans and murine neuropathy models of prediabetes and diabetes were first analyzed to identify differentially expressed TLR2- and TLR4-associated genes within the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. We observed that genes associated with TLRs 2 and 4, particularly lipopolysaccharide binding protein (LPB) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta (PIK3CB), were dysregulated across species and across multiple murine models of prediabetic and diabetic neuropathy. To further understand the role of these pathways in vivo, TLR 2 and 4 global knockout mice placed on a 60% high fat diet (HFD-TLR2/4^-/-) were compared with wild type (WT) mice on a high fat diet (HFD-WT) and WT controls on a standard diet (CON). Mice then underwent metabolic, neuropathic, and immunological phenotyping at two time points to assess the impact of TLR signaling on neuropathy and immunity during metabolic dysfunction over time. We found that HFD-TLR2/4^-/- and HFD-WT mice weighed more than CON mice but did not have increased fasting blood glucose levels. Despite normal blood glucose levels, HFD-TLR2/4^-/- mice eventually developed neuropathy at the later time point (28 wks of age) but were somewhat protected from neuropathy at the early time point (16 wks of age) as measured by shorter hind paw withdraw latencies. This is in contrast to HFD-WT mice which developed neuropathy within 11 wks of being placed on a high fat diet and were neuropathic by all measures at both the early and late time points. Finally, we immunophenotyped all three mouse groups at the later time point and found differences in the number of peripheral blood Ly6C-myeloid cells as well as F4/80+ expression. These results indicate that TLR signaling influences early development of neuropathy in sensory neurons, potentially via immune modulation and recruitment.

Keywords: High fat diet; Peripheral neuropathy; Toll-like receptor 2 and 4.

Figure 1.

Neuropathy phenotyping at early and late time points. Nerve conduction velocities (NCV; m/s) of the (A) sural and (B) sciatic-tibular motor nerves in CON (3 males and 2 females), HFD-WT (2 males and 3 females), and HFD-TLR2/4^−/− (2 males and 3 females) mice at 16 and 28 wks of age. (C) Hind paw withdrawal latency (HPL) at 16 and 28 wks of age. (D) Intraepidermal nerve fiber density (IENFD) at 28 wks of age. Data are represented as means ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Figure 2.

Peripheral blood leukocyte levels and F4/80 expression at the late time point. CON, HFD-WT, and HFD-TLR2/4^−/− mice were sacrificed at 28 wks of age, and leukocyte levels in the peripheral blood were assessed using flow cytometry (A), as was F4/80 surface expression on Ly6C− and Ly6C+ monocytes (B). Data are represented as means ± SEM. * P ≤ 0.05, ** P ≥ 0.01, and **** P ≥ 0.0001.