Sporadic ALS is characterized by large numbers of activated microglia/macrophages in the regions of the spinal cord undergoing neurodegeneration (Graves et al., 2004
; Henkel et al., 2004
). Recent studies in ALS disease models have implicated important roles for activated microglia/macrophages in ALS disease progression in vivo
(Beers et al., 2006
; Boillee et al., 2006
). sALS is also characterized by elevated levels of abnormally activated monocyte/macrophages in peripheral blood (Zhang et al., 2005
). In the current study we found that short-term cultivation of peripheral blood mononuclear cells from patients with sALS resulted in induction of genes related to the LPS/TLR4 signal transduction pathways. The pattern of gene transcription observed in sALS patients was best replicated by exposure of PBMCs from healthy subjects to the TLR4 ligand LPS, which stimulated expression of all sALS associated RNAs. Therefore, abnormally activated monocytes in sALS patients appear to be primed to become activated via LPS/TLR4 pathways. To our knowledge, this is the first demonstration of a role for LPS-mediated stimulation of the innate immune response in patients with sporadic ALS.
The monocyte ALS gene expression profile was only observed after short-term cultivation, but not in the peripheral blood mononuclear cells immediately after Percoll separation. This activation profile may be related to LPS priming of monocytes which upon cultivation results in LPS/TLR4-mediated signaling. However, if the LPS responsible for this LPS/TLR4 mediated signaling is already bound to the blood cells in vivo
, why was there not a partial stimulation of the characteristic genes in time zero samples? Certainly there is some level of abnormal monocyte activation in ALS patient blood as the level of CD14/DR expression is elevated and the degree of this elevation is related to plasma LPS levels (Zhang et al., 2009
). The LPS/TLR4 stimulation pathway induces potent responses and results in abnormally activated monocyte/macrophages and expressions of proinflammatory cytokines in vivo
(Beutler et al., 1985
; Danner et al., 1991
; Okusawa et al., 1988
; Tracey et al., 1986
); however, the systemic activation and inflammation responses may trigger the negative regulation of the TLR4 signaling pathway (Lu et al., 2008
) to down-regulate the activation of LPS/TLR4-mediated signaling which serves as a feedback or protective mechanism in modulation of inflammation-induced damage. Therefore, interferon and protein secretion associated genes may require more stimulation than is possible in the blood to see elevation of these genes. The priming of abnormally activated monocytes may also be LPS-independent, and driven by other stimuli of TLR4 signaling pathways (Biragyn et al., 2002
; Ohashi et al., 2000
; Termeer et al., 2002
), it may have to do with an unknown, ALS related process inherent to the macrophages. Further studies looking at the persistence of RNA induction in ALS will be required to test this.
Recently, HIV infected individuals with acquired immune deficiency syndrome (AIDS) have been reported to have elevations in plasma LPS levels with concomitant immune activation (Brenchley et al., 2006
). The elevated plasma LPS is associated with the gut associated microbial translocation. Increased expression of many of the genes that were significantly upregulated in this study have also been described in a study of frontal-lobe gene expression in simian immunodeficiency virus mediated encephalitis (SIVE) (Roberts et al., 2003
) which is used as a model system to understand HIV associated dementia (HAD). Our previous finding of higher levels of plasma LPS, and that increased LPS levels correlated with degree of activated monocyte-associated signaling genes in the current studies in ALS suggests that chronic stimulation of innate immunity via LPS/TLR4 signaling pathways may be a common theme in a subset of neurodegenerative diseases.
Activation of LPS/TLR4 related pathways was observed in sALS patient blood cells at all time points and independent of symptomatic phase of their disease course. Overall, approximately 90% of the samples from sALS patients exhibited elevated transcription of the LPS/TLR4 stimulated genes versus less than 5% of those from healthy individuals. In addition, the increase in RNA signals from ORM1 was associated with ALS disease severity as defined by ALSFRS-R score. Our recent studies on blood specimens from patients with sALS found elevated levels of plasma LPS, and plasma LPS levels were found to positively correlate with levels of abnormally activated monocyte/macrophages in sALS (Zhang et al., 2009
). Elevated levels of TNF-α and other pro-inflammatory cytokines have been seen in SOD1 transgenic mice (Hensley et al., 2003
) and patients with ALS (Babu et al., 2008
; Cereda et al., 2008
; Ono et al., 2001
; Poloni et al., 2000
). Chronic stimulation of innate immunity with LPS has also been shown to accelerate the disease course in SOD1 transgenic mice (Nguyen et al., 2004
). In the current study, the positive correlations between those two systemic immune parameters, degree of abnormally activated monocyte/macrophages and plasma LPS levels, and RNA signals of multiple LPS/TLR4 stimulated genes in ALS patients may provide a link between the in vitro
transcriptional response observed and the systemic immune activation, suggesting that chronic activation of monocyte/macrophages via LPS/TLR4 signaling pathways would have a deleterious effect on disease progression in ALS. Whether this PBMC sALS transcriptional response can be used as a means to identify or monitor disease progression in ALS is still an open question. Protein expression levels of those “ALS monocyte-associated response genes”, for example ORM1 protein, appear definitely worthy of further investigation. Furthermore, agents that inhibit LPS/TLR4 mediated activation would be predicted to interfere with disease progression. These observations may provide a new area of investigation for the development of novel therapeutics for ALS and other neurodegenerative diseases.