We observed a prominent procaspase 4/5 band and a procaspase 4 band with two different antibodies in human iPS-Mg exposed to FG. This expression was not observed following exposure to LPS or in untreated cells. We also did not observe an obvious cleaved product for caspase-4 but were able to observe a slight band at around p20 which is indicative of the cleaved product of active caspase4/5, again only in cells treated with FG. In rat microglia in which significant expression of procaspase-12 (the alleged homologue of caspase-4, Hitomi et al., 2004) was present in unstimulated cells as well as following FG exposure and the minor active caspase band is only evident in the FG treated rat microglia (Piers et al., 2018). It could be argued that the band in all lanes with the caspase-4/5 blots is a pro-caspase, but this was not present with the caspase-4 blot. In humans, caspase-4 is purportedly upstream and linked to the activation of the inflammasome, subsequent caspase-1 activation and the secretion of mature IL-1β. We previously observed caspase-1 activation and IL-1β secretion following direct stimulation of TREM2 in Cv iPS-Mg but reduced inflammasome activation in TREM2R47H variants (Cosker et al., 2021) indicating firstly, the ability to activate the inflammasome in human iPS-Mg and secondly, an inflammasome deficit in TREM2R47H expressing iPS-Mg. Here however, FG upregulation of procaspase 4/5 did not result in classical inflammasome activation (as evidenced by the lack of GRP78 expression, an ER chaperone heat shock protein linked to ER stress (Lee, 2005; Ibrahim et al., 2019) as well as a lack of caspase-1 activity and no IL-1β secretion. As a positive control for ER stress, thapsigargin induced an increase in GRP78 expression and caspase-1 activity, suggesting that the increased Caspase 4/5 expression we see in response to FG is not linked to canonical ER stress pathways. This was supported by our finding that caspase-1 activity was not enhanced by FG in any of the TREM2 variants, even when coupled with ATP priming of the inflammasome (Cosker et al., 2021) but was observed with LPS and ATP across all the variants. Interestingly the ER stressor thapsigargin was able to induce caspase-1 activity across all the TREM2 lines suggesting that the cellular “machinery” for ER stress pathway activation is viable in all these TREM2 variants. These data for the lack of canonical inflammasome activity by FG differ from recent findings in rodent cells (BV2 and primary rodent microglia) (Roseborough et al., 2023) and point to further differences between human and rodent cell responses.
Furthermore, the finding that NFκB phosphorylation was enhanced with FG in the Cv line but not in the TREM2R47H line or the TREM2−/− line points to differences in their abilities to activate the inflammasome as we previously reported (Cosker et al., 2021) but also suggests a non-canonical inflammasome pathway is activated by FG which does not involve caspase-1 activity or IL-1β secretion.
As NFκB is a major sensor of metabolic imbalances that result in inflammasome activation we investigated whether FG was able to induce a metabolic switch in microglia by enhancing glycolysis as we previously reported (Piers et al., 2020). Unlike LPS, FG did not induce an enhancement in glycolysis and is further evidence that FG-mediated cell signalling pathways are distinct from those induced with LPS. Furthermore, the LPS increased expression of NFκB is not due to inflammasome activity because we have not primed the cells with ATP. However, interestingly FG enhanced oxidative phosphorylation, thus differing again from LPS stimulation.
The differences we observed with cytokine secretion following FG or LPS stimulation point to differences in pathway activation following these two stimulants. Previously, we found that CXCL1, IL-6 and TNFα showed differential expression in response to LPS or FG exposure in rat primary microglia (Piers et al., 2018). In iPS-Mg, IL-1β was not significantly secreted above basal in response to FG compared with LPS. Similar findings were observed for TNFα, and IL-6. Additionally, analysis of the proteome array data indicated adiponectin was significantly elevated in response to FG; this protein is thought to limit an inflammatory microglial phenotype and promote a protective microglial phenotype through an AdipoR/NFκB pathway (Nicholas et al., 2017), which may also link with our finding of NFκB activation with FG. Complement component C5/C5a was also elevated in supernatants from iPS-Mg exposed to FG compared with LPS stimulation and comprises part of the complement pathway; C5a is a cleavage product of C5 and may serve as a microglial chemotactic agent by activating a microglial potassium current, as well as acting as an anaphylatoxin (Ilschner et al., 1996).
The finding that the CD11b receptor (also known as the MAC-1/CR3 receptor), the putative integrin class receptor for FG, (Adams et al., 2007) was not significantly upregulated in Cv iPS-Mg differs from our previous findings for rodent microglia (Piers et al., 2011). FG does not directly bind to CD11b but the cleaved product of FG, fibrin, does, and requires the presence of thrombin for this function. This supports an interpretation of our results as due to FG and not fibrin. We also showed previously that FG and fibrin have differential effects on rodent microglia and neurons (Piers et al., 2011, 2018). Additionally, FG is detected in MS brain lesions, in AD, and following traumatic brain injury (Jenkins et al., 2018, review Wen & Zhang 2023) and thus whilst some of it may be cleared by fibrinolysis, FG can persist and promote innate immune activation and local inflammation to directly damage the brain (McLarnon, 2022).
ER stress has been shown to be an important activator of the inflammasome (Li et al., 2020) which in turn has been shown to be activated in a number of neurodegenerative diseases including AD (Ravichandran & Heneka 2021) and MS (Govindarajan et al., 2020). We have also previously shown that the inflammasome can be activated in human iPS-Mg by exposure to LPS and ATP (Cosker et al., 2021). The triggering of inflammasome activation has also been proposed to occur following exposure of microglia to amyloid-β and tau oligomers in AD, α-synuclein in PD, and superoxide dismutase (SOD1) and TAR DNA-binding protein 43 (TDP43) in ALS (Ravichandran & Heneka 2021). Here FG enhanced the expression of procaspase 4/5 but did not induce caspase-1 activity or promote a glycolytic switch. LPS did induce caspase-1 activity and IL-1β secretion, as well as enhancing glycolysis, although no differences were detected between FG and LPS in terms of increases in the basal or maximal respiration. Furthermore, whilst it has been reported that FG can increase phagocytosis in mouse models (Davalos et al., 2019), here the phagocytosis of oligomeric Aβ1 − 42 by FG was reduced and our data suggest that the reduced Abeta phagocytosis we observed in the presence of FG is due to direct binding of Aβ by FG. FG can bind Aβ in monomeric and with greater affinity, oligomeric form (Ahn et al., 2014), the latter being the Aβ form used in our current study. This in turn may inhibit its phagocytosis by microglia, and since the oligomeric form of Aβ is claimed to be the most toxic species (Cleary et al., 2005), this may have consequences for Aβ clearance by microglia, toxicity to neighbouring cells and enhancement of amyloid deposition.
Thus, here we show that FG has a direct effect in a human microglial model, inducing upregulated expression of caspase 4 and caspase 4/5. In human monocytes, caspases 4 and 5 are involved in LPS-induced activation of a non-canonical inflammasome pathway (Vigano et al., 2015) whilst human caspase-4 has been shown to mediate non-canonical inflammasome activation following human macrophage exposure to gram-negative bacterial pathogens (eg Legionella pneumophila, Yersinia pseudotuberculosis, and Salmonella enterica serovar Typhimurium) (Casson et al., 2015). The data from Vigano et al., (2015) differ from ours in that procaspase 4 was found to be expressed in non-activated cells and enhanced with LPS stimulation. We did not detect any procaspase 4 expressed in our human iPS-Mg at basal or in response to LPS, although we did detect an upregulation of procaspase expression with FG. Interestingly, as with our data, these authors were unable to detect the active caspase 4 p20 SU following stimulation, whilst their use of a caspase-5 antibody did show active caspase 5 p20 SU as was detected with our use of a caspase 4/5 antibody.
Taken together, our data suggest FG activates microglia in different ways to the responses in rat microglia. Furthermore, we report here differences in the responses of iPS-Mg to FG compared with exposure to the TLR4 agonist LPS, with the activation of canonical inflammasome pathways evident with LPS but not FG. We detected minor differences between the responses of the different TREM2 variants, which may be important for microglial responses in neurodegenerative diseases where bbb dysfunction is implicated. Small but differential responses in Cv and TREM2-variant expressing Mg linked to LOAD may enhance risk. Interestingly our finding that FG binding with Aβ inhibited microglia Aβ phagocytosis may be of significance to enhanced parenchymal Aβ deposition in AD and is the first report of its inhibiting microglial phagocytosis.