Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury—Time-Dependent Changes in the Cortex, Striatum, Thalamus and Hippocampus in a Mouse Model

Abstract

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein—GFAP), microglia/macrophages (allograft inflammatory factor 1—IBA-1), and microglia (transmembrane protein 119—TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Protein Expression of the Microglial Marker Tmem119 Decreases in Association With Morphological Changes and Location in a Mouse Model of Traumatic Brain Injury

“…While we observed a drop of Tmem119 protein levels, Tmem119 gene expression increased in the contused area, with a micro topical distribution of its overexpression, mirroring that of C1q, an inflammatory gene associated with microglia activation and synaptic pruning after TBI (Krukowski et al, 2018;Ciechanowska et al, 2021). Overexpression of Tmem119 was previously reported after 4 days, and to a minor extent, 7 days after TBI in RNA extracts of the cortex, striatum, and hippocampus ipsilateral to the contusion, along with the overexpression of a wide spectrum of cellular markers for neutrophils, CD8+ T cells, astroglia, and microglia/macrophages (Ciechanowska et al, 2021). The observation that Tmem119 protein levels clearly decreased in active microglia in the TBI brain, while the mRNA increased could be due either to the impaired Tmem119 translation, possibly due to posttranscriptional modifications causing reduced Tmem119 transcript stability, or to the enhanced protein degradation.…”

Chronic complement dysregulation drives neuroinflammation after traumatic brain injury: a transcriptomic study

“…Specifically, an analysis of complement genes extracted from an RNAseq dataset acquired from the cortex at 1 day post CCI (GSE79441) [ 45 ], revealed upregulation of C1qa-c , C3ar1 , Itgam, Itgax , Cd44 , CD93 , Ager and Serping1 , and downregulation of regulatory Csmd1 , which is in line with our data. Moreover, a recent qRT-PCR study characterizing the regional expression of classical and lectin initiators up to 5 weeks after CCI reported the upregulation of C1q , C1s , and Fcnb , and the absence of MBL dysregulation [ 4 ].…”

“…Previous high-throughput studies have analyzed gene and protein expression in various experimental models of TBI, both acutely and chronically after injury [ 4 – 7 ]. Notably, local and systemic changes in the level of complement system components were consistent and of high magnitude across the various injury models at specified time points.…”

Identification of Key Genes and Pathways in the Hippocampus after Traumatic Brain Injury: Bioinformatics Analysis and Experimental Validation

“…One recent clinical study showed that elevated serum C1q levels were significantly correlated with traumatic severity and could serve as an independent prognostic factor for long-term outcomes after TBI [73]. After TBI, microglia and neurons were the primary source of C1q [74,75]. The increased expression of C1q might lead to neuron loss and chronic neuroinflammation and correlate with sleep spindle loss and epileptic spikes.…”