To identify potential inflammatory effects of PBMCs on cortical and hippocampal cells, OCHSCs from individual rats were co-cultured with PBMCs extracted from the same animal. Before adding PBMCs to OCHSCs, we incubated PBMCs with the specific TLR4 ligand LPS (signal 1) (14), followed by treatment with the fungal-derived ionophore Nigericin, which induces K+ efflux (signal 2) to activate NLRP3 (9, 14), therefore, giving rise to NLRP3 inflammasome. PBMCs were divided into three groups: 1) PBMCs stimulated by sequential incubation of LPS and Nigericin, hereafter referred to as immunoreactive PBMCs (IR-PBMCs), 2) primed PBMCs stimulated by LPS only (LPS-PBMCs), and untreated PBMCs (control PBMCs). OCHSC-PBMC co-culture groups were compared to naive OCHSCs (Fig. 1). Since different peripheral cell types can penetrate the blood-brain barrier and have the capacity to induce neuroinflammation upon extravasation (4, 5, 20, 21), we opted to include all splenic PBMC subtypes in our study rather than using a subset of purified cell types.
We first investigated the efficacy of the OCHSC-PBMC co-culture model by evaluating potential PBMC invasion of cortical and hippocampal layers upon seeding of PBMCs on top of cortical layer I of OCHSCs. Using pre-labeled control PBMCs with the fluorescent CFSE dye, we traced transmigration of PBMCs 2 hours after seeding them onto OCHSCs (Fig. 2). Transmigration of CFSE-labeled (CFSE+) cells was detected throughout the entire slice, spreading across different cortical (Fig. 2C and E), and hippocampal layers (Fig. 2C and F). Further, localization of PBMCs was not limited to superficial sections of OCHSCs, but cells were also found in deep tissue sections irrespective to the OCHSC region, thus, suggestive of the formation of cell-adhesion between PBMCs and neural cells.
We next exploited the semi-porous properties of the membrane insert, which is at an interface between OCHSCs and culture media, to assess the effect of cytokine infiltration on neuronal function independently from PBMC trafficking. We compared a) adding CFSE+ PBMCs on top of OCHSCs, where PBMCs are in direct contact with brain-derived tissue, to b) wherein CFSE+ PBMCs were added solely to the media. In contrast to OCHSC-PBMC direct co-culture, no fluorescent cells were observed in OCHSCs when PBMCs were added to the media (Fig. 2D, G and H), thus showing that the membrane is impervious to PBMCs, and appears to effectively recapitulate some aspects of the blood-brain barrier by protecting OCHSCs from PBMC infiltration.
Transduction of peripheral inflammation to neural tissue is coupled with hyperexcitability in pyramidal neurons
To address whether peripheral inflammasomes transduced to OCHSCs may give rise to functional changes in cortical and hippocampal neurons, we measured neuronal excitability by performing whole-cell electrophysiological recordings in pyramidal neurons in cortical layer II/III and in the CA1 region of the hippocampus.
Cortical pyramidal neurons from all experimental PBMC-OCHSC groups showed similar resting membrane potential values to naive OCHSCs (naive OCHSCs: -65.28 ± 6.87 mV, n = 11; control PBMCs: -68.52 ± 6.15 mV, n = 11; LPS-PBMCs: -65.25 ± 5.16 mV, n = 10; IR-PBMCs: -66.64 ± 6.75 mV, n = 10; p = 0.59; one-way ANOVA). Also, there was no change in cortical cell membrane capacitance (Cm) between OCHSCs incubated with all PBMC groups and naive OCHSCs (naive OCHSCs: 70.48 ± 13.54 pF, n = 11; control PBMCs: 70.40 ± 11.78 pF, n = 11; LPS-PBMCs: 73.04 ± 13.25 pF, n = 10; IR-PBMCs: 75.85 ± 7.55 pF, n = 10; p = 0.69; one-way ANOVA). In addition, no change in the duration of action potential repolarization (APR) was exhibited by cortical neurons in any of the PBMC-OCHSC co-cultures versus naive OCHSCs (IR-PBMCs: 2.02 ± 0.45 msec, n = 10; LPS-PBMCs: 2.11 ± 0.43 msec, n = 10; control PBMCs: 2.18 ± 0.38 msec, n = 11; naive OCHSCs: 2.07 ± 0.34 msec, n = 11; p = 0.82; one-way ANOVA) (Fig. 3Q and R). However, compared to naive OCHSCs, adding IR-PBMCs and LPS-PBMCs to OCHSCs significantly increased the input resistance (Rin) of cortical neurons, while there was no change after adding control PBMCs (naive OCHSCs: 140.35 ± 21.37 MΩ, n = 11, control PBMCs: 144.50 ± 30.92 MΩ, n = 11, p > 0.99, LPS-PBMCs: 174.20 ± 24.87 MΩ, n = 10, p < 0.05, IR-PBMCs: 180.65 ± 26.00 MΩ, n = 10, p < 0.01; one-way ANOVA). Increase of Rin manifested after adding IR-PBMCs was also significantly higher than that measured after adding control PBMCs (p < 0.05; one-way ANOVA) (Fig. 3M and N). Furthermore, while the rheobase of cortical neurons was significantly lower in the IR-PBMC-OCHSC co-culture group versus naive OCHSC and control PBMCs groups, the rheobase of cortical neurons after adding LPS-PBMCs and control PBMCs was comparable to those of naive OCHSCs (naive OCHSCs: 301.48 ± 119.33 pA, n = 11, control PBMCs: 282.36 ± 68.85 pA, n = 11; p > 0.99, LPS-PBMCs: 268.21 ± 70.90 pA, n = 10; p > 0.99, IR-PBMCs: 175.20 ± 69.12 pA, n = 10, p < 0.05 compared to naive OCHSCs, p < 0.05 compared to control PBMCs; one-way ANOVA) (Fig. 3O and P).
Hence, following the application of control, LPS- or IR-PBMCs, cortical neurons did not display noticeable electrophysiological alterations with respect to resting membrane potential, Cm or APR. Yet, co-culturing of LPS- and IR-PBMCs did induce Rin changes in cortical neurons. Interestingly, only the addition of IR-PBMCs produced changes in action potential firing propensity, i.e. rheobase, whereas the addition of LPS-primed PBMCs had no effect on cell firing.
Recordings from hippocampal pyramidal neurons were, for the most part, analogous to their cortical counterparts, since there were no notable differences between naive OCHSCs and OCHSCs co-cultured with any of the PBMC groups with respect to resting membrane potential (naive OCHSCs: -59.41 ± 3.47 mV, n = 23; control PBMCs: -59.73 ± 1.92 mV, n = 21; LPS-PBMCs: -58.85 ± 3.00 mV, n = 19; IR-PBMCs: -59.01 ± 2.49 mV, n = 20, p = 0.75; one-way ANOVA), or Cm (naive OCHSCs: 100.1 ± 20.53 pF, n = 23; control PBMCs: 100.4 ± 28.84 pF, n = 21; LPS-PBMCs: 90.69 ± 23.93 pF, n = 19; IR-PBMCs: 95.03 ± 18.44 pF, n = 20, p = 0.50; one-way ANOVA). Also, similar to cortical neurons, hippocampal neurons exhibited significantly augmented Rin after seeding of IR-PBMCs and LPS-PBMCs on OCHSCs relative to naive OCHSCs, and this augmentation was significant for both groups when compared to control PBMCs, which was not significantly different from naive OCHSCs (naive OCHSCs: 101.87 ± 9.07 MΩ, n = 23, control PBMCs: 101.87 ± 11.23 MΩ, n = 21, p > 0.99, LPS-PBMCs: 125.18 ± 11.60 MΩ, n = 19, p < 0.001 versus naive OCHSCs, p < 0.001 versus control PBMCs, IR-PBMCs: 121.46 ± 11.41 MΩ, n = 20, p < 0.001 versus naive OCHSCs, p < 0.001 versus control PBMCs; one-way ANOVA) (Fig. 4M and N).
In terms of cell firing, application of IR-PBMCs to OCHSCs resulted in rheobase reduction versus control PBMCs and naive OCHSCs. Also, there was no significant difference between control PBMC-OCHSC co-cultures and naive OCHSC. Conversely, unlike cortical pyramidal neurons, following LPS-PBMCs application, the rheobase of hippocampal neurons was markedly reduced compared to control PBMCs and naive OCHSCs (naive OCHSCs: 261.77 ± 80.86 pA, control PBMCs: 261.01 ± 46.13 pA, n = 21, p > 0.99, LPS-PBMCs: 194.92 ± 61.14 pA, n = 19, p < 0.01 versus naive OCHSCs, p < 0.01 versus control PBMCs, IR-PBMCs: 191.96 ± 48.96, n = 20, p < 0.01 versus naive OCHSCs, p < 0.01 versus control PBMCs; one-way ANOVA) (Fig. 4O and P). Thus, in contrast to the cortex, the effect of LPS-primed PBMCs on rheobase in the hippocampus was reminiscent to IR-PBMCs, eliciting hyperexcitability in OCHSCs co-cultured with LPS-PBMCs and IR-PBMCs versus the control groups.
Likewise, the repolarization phase was notably extended in hippocampal pyramidal neurons recorded following IR-PBMCs and LPS-PBMCs incubation, when compared to control PBMCs incubation and naive OCHSCs, with no significant change between naive OCHSCs and OCHSCs co-cultured with control PBMCs (naive OCHSCs: 1.31 ± 0.16 msec, n = 15, control PBMCs: 1.29 ± 0.20 msec, n = 15, p > 0.99, LPS-PBMCs: 1.53 ± 0.20 msec, n = 15, p < 0.05 versus naive OCHSCs, p < 0.05 versus control PBMCs, IR-PBMCs: 1.52 ± 0.20 msec, n = 15, p < 0.05 versus naive OCHSCs, p < 0.01 versus control PBMCs; one-way ANOVA) (Fig. 4Q and R). Accordingly, unlike the cortex where none of the PBMC groups had an effect on APR, LPS- and IR-PBMCs generated extended APR in hippocampal neurons, suggestive of different action potential kinetics between cortical and hippocampal neurons.
Taken together, upon application on OCHSCs, both LPS- and IR-PBMCs altered some of the intrinsic properties of cortical and hippocampal pyramidal neurons associated with excitability. Notably, enhancement of neuronal excitability appears to be concomitant with the expression of NLRP1 and/or NLRP3 inflammatory markers in OCHSCs.
Activation of inflammasomes in OCHSCs promotes a decline in 4-AP sensitive transient K + currents in pyramidal neurons
Reduction of rheobase and prolongation of the repolarization duration in pyramidal neurons after exposure to PBMCs is likely to arise from changes in the properties of specific ion channels that are activated when graded potential reaches threshold potential as well as during the repolarization phase. Thus, we next asked which ion channels would satisfy these criteria.
Interestingly, transient A-type fast activating, fast inactivating voltage-dependent K+ current (IA) is known to regulate action potential rheobase and waveform (24–26). In addition, LPS incubation with caudate nucleus cultures has been shown to reduce IA in vitro (27). Therefore, we postulated that suppression of IA might be a plausible culprit underlying neuronal excitability changes elicited by inflammation. To explore putative suppression of IA in OCHSCs, we selectively inhibited IA by adding 3 mM of 4-AP during recording of pyramidal neurons. Our approach was to distinguish IA from other currents initiated at threshold potential and activated throughout different action potential phases. Hence, we obtained whole-cell recordings while blocking Na+, Ca2+ currents together with the majority of K+ currents (also referred to as baseline conditions), then repeated the same recordings again while adding 4-AP to the abovementioned current blockers, and derived net IA by calculating current difference between both conditions.
IA in cortical neurons was notably abrogated in the IR-PBMCs group versus naive OCHSCs and control PBMCs groups, whereas cortical neurons in the LPS-PBMCs and control PBMCs groups showed similar IA activation to naive OCHSCs (current response at + 20 mV, i.e. maximum voltage input, naive OCHSCs: 897.53 ± 87.52 pA, n = 6, control PBMCs: 972.12 ± 129.91 pA, n = 6, p > 0.99, LPS-PBMCs: 791.12 ± 116.90 pA, n = 6, p = 0.82, IR-PBMCs: 515.81 ± 58.65 pA, n = 6, p < 0.01 versus naive OCHSCs, p < 0.001 versus control PBMCs, repeated measures one-way ANOVA) (Fig. 5A, B and E). On the other hand, adding either IR-PBMCs or LPS-PBMCs to OCHSCs substantially reduced IA in hippocampal neurons relative to naive OCHSCs and to control PBMCs. Markedly, the effect of co-culturing control PBMCs with OCHSCs was opposite to LPS- and IR-PBMCs, resulting in potentiation of IA amplitude compared to naive OCHSCs (current response at + 20 mV, naive OCHSCs: 928.82 ± 83.55 pA, n = 7, control PBMCs: 930.35 ± 145.81 pA, n = 6, p < 0.0001, LPS-PBMCs: 648.57 ± 111.67 pA, n = 6, p < 0.0001 versus naive OCHSCs, p < 0.0001 versus control PBMCs, IR-PBMCs: 617.99 ± 52.41 pA, n = 6, p < 0.001 versus naive OCHSCs, p < 0.0001 versus control PBMCs, repeated measures one-way ANOVA) (Fig. 5C, D and G).
Beside IA, we also considered other potential ion channel candidates influenced by PBMC-induced neuroinflammation. Given that transient D-type fast activating, slowly inactivating voltage-dependent K+ current (ID) is sensitive to 4-AP much like IA, and activation of ID delays action potential firing along with reducing action potential duration in pyramidal neurons (24, 26, 28, 29), we decided to investigate the possible contribution of ID in inflammation-induced excitability. However, while 4-AP inhibits IA activation in millimolar concentrations, (1–3 mM), ID is highly sensitive to 4-AP, i.e. in micromolar concentrations (30–100 µM) (24–26, 28). Therefore, we isolated ID using the same approach as IA, except the concentration of 4-AP was 40 µM, instead of 3 mM.
Results from pyramidal neurons in the cortex revealed that relative to naive OCHSCs, the amplitude of ID was significantly diminished only in the IR-PBMCs co-cultured with OCHSCs, whereas there were no notable changes in ID amplitude upon co-culturing control PBMCs or LPS-PBMCs with OCHSCs (current response at + 20 mV, naive OCHSCs: 256.25 ± 15.01, n = 9, control PBMCs: 226.92 ± 10.84 pA, n = 8, p = 0.08, LPS-PBMCs: 206.64 ± 24.01 pA, n = 9, p = 0.35, IR-PBMCs: 175.03 ± 23.88 pA, n = 9, p < 0.01, repeated measures one-way ANOVA) (Fig. 5H, I and L). In hippocampal neurons, OCHSCs co-cultured with IR-PBMCs and LPS-PBMCs manifested significantly lower ID than naive OCHSCs as well as control PBMC-OCHSC co-cultures, whereas adding control PBMCs did not result in significant changes in ID compared to naive OCHSCs (current response at + 20 mV, naive OCHSCs: 253.39 ± 21.49 pA, n = 11, control PBMCs: 229.38 ± 20.04 pA, n = 12, p = 0.06, LPS-PBMCs: 161.32 ± 15.81 pA, n = 12, p < 0.0001 versus naive OCHSCs, p < 0.001 versus control PBMCs, IR-PBMCs: 155.66 ± 15.74 pA, n = 11, p < 0.0001 versus naive OCHSCs, p < 0.0001 versus control PBMCs, repeated measures one-way ANOVA) (Fig. 5J, K and N).
Collectively, changes in IA and ID are in line with our previous data, where decay of IA and ID was only detected in pyramidal neurons that exhibited changes in cell firing associated with inflammasome induction, i.e. the cortex of IR-PBMC-OCHSC co-cultures and the hippocampus of IR-PBMC- and LPS-PBMC-OCHSC co-cultures. Thus, transduction of peripheral inflammation to OCHSCs leads to a decrease in the amplitude of IA and ID, which consequently promotes hyperexcitability in pyramidal neurons.
Direct cell-cell contact between PBMCs and OCHSCs is required for pro-inflammatory signal transduction from LPS-primed PBMCs to brain-derived tissue
To gain further insight into how pro-inflammatory PBMCs can transduce peripheral inflammation to OCHSCs, we investigated whether diffusible factors secreted by PBMCs, primarily cytokines and chemokines, can mediate inflammasome transduction solely by binding to their cognate receptors found in OCHSC cells (17). Since we have demonstrated that when PBMCs were added to the media, the membrane insert acts as a barrier by preventing cell-cell adhesion between PBMCs and OCHSCs (see Sect. 4.1), we decided to add PBMCs to OCHSC media, where presumably PBMC-derived cytokines and chemokines, but not PBMCs themselves, can diffuse through the membrane. To inquire whether the outcome of adding PBMCs to OCHSC media would mimic that of co-culturing PBMCs in direct contact with OCHSCs, we examined potential expression of NLRP1 and NLRP3 inflammasome markers along with potential changes in excitability parameters and 4-AP sensitive currents after adding control PBMCs, LPS-PBMCs and IR-PBMCs to the media. In the next set of experiments, we only investigated the effects of media-incubated PBMCs on the hippocampus because the hippocampus was more susceptible to PBMC-induced hyperexcitability than the cortex.
Our immunohistochemical analysis of the inflammasome markers NLRP1, NLPR3 and the downstream effector ASC in OCHSCs revealed that media incubation of IR-PBMCs (mIR-PBMCs) gave rise to the upregulation of NLRP1 (Fig. 6G), NLRP3 (Fig. 6H) and ASC (Fig. 6I and I´) in the hippocampus. However, there was no detectable expression of NLRP1, NLRP3 or ASC following media incubation of LPS-PBMCs (mLPS-PBMCs) (Fig. 6D-F´) as compared to control PBMCs (mCTL-PBMCs) (Fig. 6A-C´). The presence of inflammasomes in the mIR-PBMCs group supports the notion that IR-PBMCs can transduce inflammation through diffusible cytokines, whereas failure of mLPS-PBMCs to evoke similar effect suggests that LPS-PBMCs require cell-cell contact for inflammation transduction.
Also, following media incubation of all experimental PBMC groups, the values of Rin, rheobase and APR in hippocampal pyramidal neurons exposed to mLPS-PBMCs were reminiscent to hippocampal neurons exposed to mCTL-PBMCs, and consistent with the absence of hippocampal inflammation in OCHSCs following mLPS-PBMCs. However, in hippocampal neurons exposed to mIR-PBMCs, Rin, rheobase and APR values were significantly different from mCTL-PBMCs, again consistent with inflammasome induction in OCHSCs from this group (Rin, mCTL-PBMCs: 109.30 ± 12.98 MΩ, n = 11, mLPS-PBMCs: 117.78 ± 15.50 MΩ, n = 11, p = 0.41, mIR-PBMCs: 127.51 ± 9.64 MΩ, n = 10, p < 0.01; rheobase, mCTL-PBMCs: 212.63 ± 46.88 pA, mLPS-PBMCs: 192.86 ± 36.64 pA, n = 11, p = 0.93, mIR-PBMCs: 155.55 ± 50.95 pA, n = 10, p < 0.05; APR, mCTL-PBMCs: 1.31 ± 0.14 msec, n = 11, mLPS-PBMCs: 1.36 ± 0.17 msec, n = 11, p > 0.99, mIR-PBMCs: 1.52 ± 0.20 msec, n = 10, p < 0.05; one-way ANOVA) (Fig. 6J-L). Hence, in contrast to adding LPS-PBMCs on top of OCHSCs (Sect. 4.3, Fig. 4G-I´ and M-R), the outcome of mLPS-PBMCs was comparable to that of mCTL-PBMCs, indicating that media application of LPS-PBMCs occludes their influence on neuronal excitability. Importantly, mIR-PBMCs retained their capacity to trigger neuroinflammation and enhance neuronal excitability, suggesting that diffusion of inflammatory factors secreted by mIR-PBMCs, likely cytokines, rather than cell-cell contact is responsible of the phenotypic changes observed in OCHSCs.
Furthermore, the difference of the amplitude of the 4-AP sensitive IA and ID between hippocampal neurons of OCHSCs incubated with mLPS-PBMCs and those incubated with mCTL-PBMCs was not significant. Still, both IA and ID amplitudes were significantly lower in hippocampal neurons when OCHSCs were incubated with mIR-PBMCs as opposed to those incubated with mCTL-PBMCs and mLPS-PBMCs (current response at + 20 mV, IA, mCTL-PBMCs: 1178.43 ± 119.61 pA, n = 6, mLPS-PBMCs: 1100.72 ± 45.87 pA, n = 5, p > 0.99, mIR-PBMCs: 855.10 ± 66.09 pA, n = 5, p < 0.01 versus mCTL-PBMCs, p < 0.01 versus mLPS-PBMCs; ID, mCTL-PBMCs: 254.20 ± 27.69 pA, n = 8, mLPS-PBMCs: 280.10 ± 20.16 pA, n = 8, p > 0.99, mIR-PBMCs: 160.48 ± 25.92 pA, n = 7, p < 0.05 versus mCTL-PBMCs, p < 0.01 versus mLPS-PBMCs; repeated measures one-way ANOVA) (Fig. 6M-Q). The fact that significant decrease of amplitude of IA and ID was present only in the mIR-PBMCs group further confirms the correlation between PBMC-induced hyperexcitability and changes in these 4-AP sensitive currents.
In essence, with respect to the hippocampus, mIR-PBMCs replicated the effects of co-culturing IR-PBMCs with OCHSCs, reproducing neuroinflammation and ID-/IA-mediated hyperexcitability. On the contrary, mLPS-PBMCs unexpectedly failed to provoke neuroinflammation or neuronal hyperexcitability unlike LPS-PBMC-OCHSC co-cultures. Thus, our data shows that in the case of IR-PBMCs, cell-cell contact between PBMCs and OCHSCs is not necessary to yield neuroinflammation in OCHSC hippocampal cells, thereby suggesting that PBMC-derived pro-inflammatory cytokines are potential mediators of peripheral inflammatory transduction. Nevertheless, cell-cell contact seems to be essential for transducing peripheral inflammasomes from LPS-PBMCs to the hippocampus in OCHSCs, underscoring the possibility that PBMC binding to an antigen enriched in the hippocampus is necessary for immunologically-primed PBMCs to become immunologically-activated (see Discussion).
Neuroinflammation-mediated attenuation of 4-AP sensitive transient K</> + currents is dependent on Caspase-1 activation in hippocampal cells
A key regulatory step in the oligomerization of Caspase-1-activating inflammasome complexes is the cleavage of the precursor form of Caspase-1, i.e. pro-Caspase-1, into its activated form (12, 14, 22). Indeed, inhibiting Caspase-1 cleavage using the selective Caspase-1 inhibitor VX-765 has proved to be highly effective in reducing downstream activation of NLRP1 and NLRP3 inflammasomes in the CNS (30, 31).
To test if Caspase-1 inhibition could possibly mitigate PBMC-induced neuroinflammation in the hippocampus, OCHSCs were pre-treated with VX-765 one hour before seeding LPS-PBMCs and IR-PBMCs on top of OCHSCs. Thereafter, we looked into the profile expression of NLRP1 and NLRP3 inflammasome markers, and also measured excitability parameters (Rin, rheobase, APR) together with 4-AP sensitive currents following the co-culturing of LPS-PBMCs (VX765-LPS-PBMCs) and IR-PBMCs (VX765-IR-PBMCs) with OCHSCs. Because VX-765 was dissolved in DMSO, we pre-treated OCHSCs with DMSO, i.e. the vehicle, one hour prior to applying LPS-PBMCs (DMSO-LPS-PBMCs) and deemed this group as the positive control group.
We found augmented NLRP1, NLRP3 and ASC immunolabeling in the DMSO-LPS-PBMCs group (Fig. 7A-C´) as opposed to VX765-LPS-PBMCs (Fig. 7D-F´) and VX765-IR-PBMCs (Fig. 7G-I´). Thus, these observations implicate VX-765 as a potent inhibitor of PBMC-transduced inflammasomes in OCHSC hippocampal tissue.
In addition, patch-clamp recordings showed that hippocampal pyramidal neurons displayed significantly lower Rin, lower rheobase and slower APR in DMSO-LPS-PBMC co-cultures relative to VX765-LPS-PBMCs and VX765-IR-PBMC co-cultures (Rin, DMSO-LPS-PBMCs: 132.44 ± 20.12 MΩ, n = 9, VX765-LPS-PBMCs: 104.20 ± 19.07 MΩ, n = 9, p < 0.01, VX765-IR-PBMCs: 110.44 ± 13.91 MΩ, n = 9, p < 0.05; rheobase, DMSO-LPS-PBMCs: 173.29 ± 37.27 pA, n = 9, VX765-LPS-PBMCs: 268.52 ± 79.76 pA, n = 9, p < 0.05, VX765-IR-PBMCs: 281.13 ± 98.83 pA, n = 9, p < 0.05; APR, DMSO-LPS-PBMCs: 1.56 ± 0.20 msec, n = 9, VX765-LPS-PBMCs: 1.32 ± 0.16 msec, n = 9, p < 0.05,VX765-IR-PBMCs: 1.31 ± 0.17 msec, n = 9, p < 0.05; one-way ANOVA) (Fig. 7J-L). Further, IA and ID amplitudes of hippocampal neurons from DMSO-LPS-PBMC co-cultures were notably reduced versus VX765-LPS-PBMCs and VX765-IR-PBMC co-cultures (current response at + 20 mV, IA, DMSO-LPS-PBMCs: 747.64 ± 64.58 pA, n = 5, VX765-LPS-PBMCs: 1080.72 ± 52.84 pA, n = 5, p < 0.001, VX765-IR-PBMCs: 983.24 ± 97.73 pA, n = 5, p < 0.01; ID, DMSO-LPS-PBMCs: 171.61 ± 11.41, n = 7, VX765-LPS-PBMCs: 266.99 ± 43.55 pA, n = 8, p < 0.001, VX765-IR-PBMCs: 236.95 ± 34.51 pA, n = 8, p < 0.05; repeated measures one-way ANOVA) (Fig. 7M-P). These electrophysiological findings were concordant with VX-765-induced suppression of peripheral inflammasome transduction to the hippocampus, and strongly suggest that IA- and ID-mediated hyperexcitability is caused by inflammasome formation.
Taken together, this data provides evidence that in the hippocampus, VX-765 treatment reversed the hyperexcitability concurrent with neuroinflammation triggered by pro-inflammatory PBMCs, therefore implicating Caspase-1–dependent pathway(s) as the main underlying mechanism of PBMC-induced IA- and ID-mediated enhanced excitability.