Etiogenic factors present in the cerebrospinal fluid from amyotrophic lateral sclerosis patients induce predominantly pro-inflammatory responses in microglia

Mishra et al. Journal of Neuroinflammation (2017) 14:251 DOI 10.1186/s12974-017-1028-x RESEARCH Open Access Etiogenic factors present in the cerebrospinal fluid from amyotrophic lateral sclerosis patients induce predominantly pro-inflammatory responses in microglia Pooja-Shree Mishra1,2, K. Vijayalakshmi1, A. Nalini3, T. N. Sathyaprabha1, B. W. Kramer4, Phalguni Anand Alladi1 and T. R. Raju1* Abstract Background: Microglial cell-associated neuroinflammation is considered as a potential contributor to the pathophysiology of sporadic amyotrophic lateral sclerosis. However, the specific role of microglia in the disease pathogenesis remains to be elucidated. Methods: We studied the activation profiles of the microglial cultures exposed to the cerebrospinal fluid from these patients which recapitulates the neurodegeneration seen in sporadic amyotrophic lateral sclerosis. This was done by investigating the morphological and functional changes including the expression levels of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), TNF-α, IL-6, IFN-γ, IL-10, inducible nitric oxide synthase (iNOS), arginase, and trophic factors. We also studied the effect of chitotriosidase, the inflammatory protein found upregulated in the cerebrospinal fluid from amyotrophic lateral sclerosis patients, on these cultures. Results: We report that the cerebrospinal fluid from amyotrophic lateral sclerosis patients could induce an early and potent response in the form of microglial activation, skewed primarily towards a pro-inflammatory profile. It was seen in the form of upregulation of the pro-inflammatory cytokines and factors including IL-6, TNF-α, iNOS, COX-2, and PGE2. Concomitantly, a downregulation of beneficial trophic factors and anti-inflammatory markers including VEGF, glial cell line-derived neurotrophic factor, and IFN-γ was seen. In addition, chitotriosidase-1 appeared to act specifically via the microglial cells. Conclusion: Our findings demonstrate that the cerebrospinal fluid from amyotrophic lateral sclerosis patients holds enough cues to induce microglial inflammatory processes as an early event, which may contribute to the neurodegeneration seen in the sporadic amyotrophic lateral sclerosis. These findings highlight the dynamic role of microglial cells in the pathogenesis of the disease, thus suggesting the need for a multidimensional and temporally guarded therapeutic approach targeting the inflammatory pathways for its treatment. Keywords: Sporadic ALS, Microglia, ALS-CSF, Neuroinflammation, Non-cell autonomous pathology, Chitotriosidase, IL-6, IL-10, TNF-α, IFN-γ, PGE2, COX-2, iNOS, Arginase, VEGF, GDNF * Correspondence: 1 Department of Neurophysiology, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore 560029, India Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mishra et al. Journal of Neuroinflammation (2017) 14:251 Background The exact pathomechanism of neurodegeneration in amyotrophic lateral sclerosis (ALS), esp. the sporadic forms of ALS, remains poorly understood, thus leading to the lack of an effective therapeutic intervention for the disease [1, 2]. Several studies have reported a non-cell autonomous glial involvement in the pathogenesis, and the relevance of innate as well as adaptive immunity in ALS has also been widely discussed [3]. However, the requirement of the dynamic modulatory signals from within the central nervous system (CNS) for the recruitment and regulation of the adaptive immunity across the blood-brain barrier (BBB)/ brain-spinal cord barrier (BSCB) further highlights the importance of the resident immune cells of the CNS in the disease pathology [4]. Microglia, the specialized immune cells of the CNS, constantly survey and dynamically regulate the neuronal milieu in the healthy and diseased CNS [5]. Depending on the nature and extent of the insult, microglial cells have been proposed to adopt morphologically and functionally distinct reactive phenotypes. These distinct phenotypes may perform diversified functions ranging from facilitating the pro-inflammatory processes that promote neuroinflammation to inducing an anti-inflammatory process that is engaged in healing and wound repair [6]. A fine balance among these phenotypes is considered to be crucial for a competent surveillance system, and its disruption could lead to the selfpropagating chronic neuroinflammation seen in several neurological disorders [7]. However, the typical classification based on microglial polarization into classically (M1) and alternatively (M2) activated microglia is controversial and has been parallelly challenged [8]. Activated microglia have previously been reported in the autopsy samples and animal models of familial ALS (FALS), as well as throughout the symptomatic stages as demonstrated by neuroimaging of the ALS patients [9–11]. While many studies report microglial activation to be actively neurotoxic in ALS [12–14], there are also studies that report the microglial involvement to ne either neuroprotective or having no significant role to play in the event of neurodegeneration in such ALS models [15, 16]. Further, the spatial microglia appeared to affect their activation status in these animal models [17]. Incidentally, majority of these studies were conducted with the transgenic models containing superoxide dismutase 1 (SOD1) mutations that are uncommon in sporadic ALS (SALS) (> 1%) [18]. In recent years, the focus has largely shifted to transgenic models with novel, gene mutations more commonly reported in ALS patients, thus narrowing the gap between animal models and Page 2 of 18 actual disease etiopathogenesis. Of prominence are ubiquitinated cytoplasmic inclusions (~ 98%)/mutations (FALS 5%, SALS 1%) in 43-kDa TAR DNAbinding protein (TDP-43), fused in sarcoma (FUS) (FALS 4%, SALS < 1%) and, most prominently, the hexaneucleotide repeat expansion in C9orf72 (FALS 40%, SALS > 10%) [19–21]. However, while these models recapitulate the etiopathogenesis of FALS more effectively, their relevance from the perspective of SALS pathogenesis, which constitutes 90% of the ALS etiology, is still dubious [22]. Although the emergence of these models has opened newer research avenues including aberrant RNA processing and protein degradation pathways, as well as perturbed nucleocytoplasmic transport [21], the neuroinfla (...truncated)