To identify potential inflammatory effects of PBMCs on cortical and hippocampal cells, OCHSCs prepared from P9/P10 rats were co-cultured with PBMCs extracted from the spleen at the same age. Since rats used in this study were obtained from an outbred rodent stock, OCHSCs and PBMCs were harvested from the same animal in order to reduce alloreactive T lymphocyte recognition of non-self major histocompatibility complex variants present in cells derived from other animals. Before adding PBMCs to OCHSCs, PBMCs were primed using the TLR4 ligand LPS (signal 1) (14), then exposed to the K+ antiporter nigericin, which depletes intracellular K+ concentration (signal 2) (9, 14), thereby fully activating the 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 3) untreated PBMCs (control PBMCs). OCHSC-PBMC co-culture groups were compared to naive OCHSCs (Fig. 1). Because different peripheral cell types can penetrate the blood-brain barrier and have the capacity to induce neuroinflammation upon extravasation (4, 5, 27, 28), 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 OCHSC, 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.
These findings denote that PBMCs retain their motility in vitro, with the capacity to crawl towards the cortex and the hippocampus upon the establishment of cell-cell contact between themselves and OCHSC tissue. Moreover, when PBMCs are applied to the media, the semi-porous membrane acts as a physical barrier isolating OCHSCs from PBMCs.
4.2. The hippocampus is more susceptible to PBMC-induced neuroinflammation than the cortex
PBMCs can transduce peripheral inflammatory signals to the CNS via secretory pro-inflammatory cytokines, such as IL-1β and IL-18, which can trigger neuroinflammation by binding to cognate receptors on the surface of neuronal and glial cells (e.g. IL-1R1 and IL-1R5) (29). Since exposure to both LPS and nigericin is required for activation of Caspase-1 (11, 12, 14), pro-inflammatory cytokine release should be restricted to the two-signal activated IR-PBMCs, whereas LPS-PBMCs (signal 1 only) and control PBMCs, lack the capacity for cytokine production. Importantly, pro-inflammatory cytokines such as IL-1β, that are released following NLRP3 inflammasome activation can in turn serve as a signal 1 to prime neighbouring IL-1R1-expressing cells (30).
To verify potential cytokine-mediated transduction of peripheral inflammation to neural tissue when PBMCs come into close proximity with OCHSCs, we used semi-quantitative immunohistochemical analysis to characterize the expression profile of inflammasome markers, such as NLRP1 and NLRP3, in cortical and hippocampal OCHSC tissue. Immunolabeling of NLRP1 revealed that adding IR-PBMCs to OCHSCs resulted in NLRP1 induction in both cortical (Fig. 3J) and hippocampal cells (Fig. 4J) compared to naive OCHSCs (cortex, Fig. 3A; hippocampus, Fig. 4A). However, this trend was significant only in the hippocampus (cortex, Fig. 3M, p = 0.08; hippocampus, Fig. 4M, p < 0.01; one-way ANOVA). Surprisingly, following the administration of LPS-PBMCs, NLRP1 was significantly upregulated in the hippocampus (Fig. 4G and M; p < 0.05; one-way ANOVA), but not in the cortex (Fig. 3G and M; p = 0.26; one-way ANOVA) of OCHSCs, suggesting that immunologically primed PBMCs are capable of transducing peripheral inflammation to hippocampal cells. Conversely, NLRP1 was absent in the cortex (Fig. 3D and M; p > 0.99; one-way ANOVA) and hippocampus (Fig. 4D and M; p > 0.99; one-way ANOVA) of OCHSCs co-cultured with control PBMCs.
Next, we analyzed NLRP3 expression in OCHSCs. Similar to NLRP1 immunostaining results, we found NLRP3 immunoreactive cells in the cortex and the hippocampus of IR-PBMC-OCHSC co-cultures (cortex, Fig. 3K and N, p < 0.001; hippocampus, Fig. 4K and N, p < 0.0001, one-way ANOVA). However, NLRP3 was markedly upregulated in both the cortex (Fig. 3H and N; p < 0.01; one-way ANOVA) and the hippocampus (Fig. 4H and N; p < 0.01; one-way ANOVA) of LPS-PBMC-OCHSC co-cultures as opposed to naive OCHSCs (cortex, Fig. 3B and N; hippocampus, Fig. 4B and N). Thus, unlike NLRP1 inflammasomes, NLRP3 inflammasomes can be activated in CNS cells upon exposure to LPS-PBMCs. On the other hand, NLRP3 was neither detectable in the cortex (Fig. 3E and N; p > 0.99; one-way ANOVA) nor in the hippocampus (Fig. 4E and N; p > 0.99; one-way ANOVA) when control PBMCs were added to OCHSCs.
A signature aspect of NLRP1 and NLRP3 inflammasome assembly and maturation is activation of the scaffolding protein ASC (31), however, NLRP1 can also promote inflammasome assembly independent of ASC by interacting directly with Caspase-1 (32). Therefore, we next investigated potential enhancement of ASC synthesis in OCHSCs following LPS- and IR-PBMCs application to OCHSCs. ASC was notably elevated in both the cortex and the hippocampus upon co-culturing OCHSCs with IR-PBMCs (cortex, Fig. 3L, L´ and O; hippocampus, 4L, L´ and O) and LPS-PBMCs (cortex, Fig. 3I, I´ and O; hippocampus, 4I, I´ and O) as compared to the cortex and hippocampus of control PBMC-OCHSC co-cultures (cortex, Fig. 3F, F´ and O; hippocampus, 4F, F´ and O) (IR-PBMCs: cortex, p < 0.01, hippocampus, p < 0.001; LPS-PBMCs: cortex, p < 0.01, hippocampus, p < 0.01, one-way ANOVA) and naive OCHSCs (cortex, Fig. 3C, C´ and O; hippocampus, Fig. 4C, C´ and O) (IR-PBMCs: cortex, p < 0.05, hippocampus, p < 0.01; LPS-PBMCs: cortex, p < 0.05, hippocampus, p < 0.05; one-way ANOVA). Hence, ASC expression was only detected when NLRP3 was also expressed.
Altogether, analysis of inflammasome induction in OCHSCs indicates that signal 1 priming of PBMCs via LPS, irrespective of signal 2 activation, was sufficient to transduce peripheral pro-inflammatory signals to OCHSCs, which is manifested by activation of inflammasome cascades in cortical and hippocampal cells. Nevertheless, expression of inflammasome proteins was more pronounced in the hippocampus than in the cortex. Additionally, adding control PBMCs to OCHSCs appears to be innocuous, as there was no NLRP1 or NLRP3 inflammasome recruitment in either the cortex or the hippocampus.
4.3. Transduction of peripheral inflammation to neural tissue is coupled with hyperexcitability in pyramidal neurons
To address whether peripheral inflammation transduced to OCHSCs gives 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, i.e. control PBMCs, LPS-PBMCs and IR-PBMCs, showed similar resting membrane potential values to naive OCHSCs (Table S2; p = 0.59, one-way ANOVA). In addition, we found no change in cortical cell membrane capacitance (Cm) between OCHSCs incubated with all PBMC groups and naive OCHSCs (Table S2; p = 0.69, one-way ANOVA) or in the duration of action potential repolarization (APR) exhibited by cortical neurons between OCHSCs incubated with all PBMC groups and naive OCHSCs (Fig. 3R and U; p = 0.82, one-way ANOVA). However, compared to naive OCHSCs, adding IR-PBMCs and LPS-PBMCs to OCHSCs significantly increased the input resistance (Rin) of cortical neurons (Fig. 3P and S; p < 0.05, p < 0.01, respectively, one-way ANOVA), whereas there was no change after adding control PBMCs (Fig. 3P and S; p > 0.99; one-way ANOVA). This increase of Rin after adding IR-PBMCs was significantly higher than that measured after adding control PBMCs (Fig. 3P and S; p < 0.05; one-way ANOVA). 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 (Fig. 3Q and T; p < 0.05 and p < 0.05, respectively, one-way ANOVA), the rheobase of cortical neurons after adding LPS-PBMCs and control PBMCs was comparable to those of naive OCHSCs (Fig. 3Q and T; p > 0.99 and p > 0.99, respectively; one-way ANOVA).
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 (Table S2; p = 0.75; one-way ANOVA), or Cm (Table S2; 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 (Fig. 4P and S; p < 0.001 and p < 0.001, respectively, one-way ANOVA) relative to naive OCHSCs, and this augmentation was significant for both groups when compared to control PBMCs (Fig. 4P and S; IR-PBMCs: p < 0.001, LPS-PBMCs: p < 0.001; one-way ANOVA), which was not significantly different from naive OCHSCs (Fig. 4P and S; p > 0.99; one-way ANOVA).
In terms of cell firing, application of IR-PBMCs to OCHSCs resulted in rheobase reduction versus control PBMCs and naive OCHSCs (Fig. 4Q and T; p < 0.01 versus naive OCHSCs, p < 0.01 versus control PBMCs, one-way ANOVA). Also, there was no significant difference between control PBMC-OCHSC co-cultures and naive OCHSC (Fig. 4Q and T; p > 0.99; one-way ANOVA). Conversely, unlike cortical pyramidal neurons, following LPS-PBMCs application, the rheobase of hippocampal neurons was markedly reduced compared to control PBMCs (Fig. 4Q and T; p < 0.01; one-way ANOVA) and naive OCHSCs (Fig. 4Q and T; p < 0.01; one-way ANOVA). Thus, in contrast to the cortex, the effect of LPS-primed PBMCs on rheobase in the hippocampus was reminiscent to IR-PBMCs, where both groups elicited comparable hyperexcitability in OCHSCs when compared to the control groups.
Likewise, APR was notably extended in hippocampal pyramidal neurons recorded following IR-PBMCs and LPS-PBMCs incubation, when compared to control PBMCs incubation and naive OCHSCs (Fig. 4R and U; IR-PBMCs: p < 0.05 versus naive OCHSCs, p < 0.05 versus control PBMCs; LPS-PBMCs: p < 0.05 versus naive OCHSCs, p < 0.01 versus control PBMCs, one-way ANOVA), with no significant change between naive OCHSCs and OCHSCs co-cultured with control PBMCs (Fig. 4R and U; p > 0.99; one-way ANOVA). Accordingly, unlike the cortex where none of the PBMC groups had any effect on APR, LPS- and IR-PBMCs generated APR extension in hippocampal neurons, which indicates 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 correlates with the expression of NLRP1 and/or NLRP3 inflammatory markers in OCHSCs.
4.4. Activation of inflammasomes in OCHSCs promotes a decline in 4-AP sensitive transient K + currents in pyramidal neurons
The 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 (33–35). In addition, LPS incubation with caudate nucleus cultures has been shown to reduce IA in vitro (36). 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 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 3 mM of 4-AP to the abovementioned current blockers to selectively inhibit IA (Fig. 5F). Net IA was derived by calculating current difference between both conditions.
IA in cortical neurons was notably abrogated in the IR-PBMCs group versus naive OCHSCs (Fig. 5A, B and E; p < 0.01; repeated measures one-way ANOVA) and control PBMCs groups (Fig. 5E; p < 0.001; repeated measures one-way ANOVA), whereas cortical neurons in the LPS-PBMCs and control PBMCs groups showed similar IA activation to naive OCHSCs (Fig. 5E; p > 0.99 and p = 0.82, respectively; repeated measures one-way ANOVA). On the other hand, adding either IR-PBMCs or LPS-PBMCs to OCHSCs substantially reduced IA in hippocampal neurons relative to naive OCHSCs (Fig. 5C, D and G; IR-PBMCs: p < 0.001; LPS-PBMCs: p < 0.0001; repeated measures one-way ANOVA) and to control PBMCs (Fig. 5G; IR-PBMCs: p < 0.0001; LPS-PBMCs: p < 0.0001; repeated measures one-way ANOVA). 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 (Fig. 5G; p < 0.0001; repeated measures one-way ANOVA).
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 (33, 35, 37, 38), 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) (33–35, 37). Therefore, we isolated ID using the same approach as IA, except the concentration of 4-AP was 40 µM, instead of 3 mM (Fig. 5M).
Results from pyramidal neurons in the cortex revealed that relative to naive OCHSCs, the amplitude of ID was significantly diminished only when OCHSCs were co-cultured with IR-PBMCs (Fig. 5H, I and L; p < 0.01; repeated measures one-way ANOVA), whereas there were no significant changes in ID amplitude upon co-culturing OCHSCs with control PBMCs (Fig. 5H; p = 0.08) ; repeated measures one-way ANOVA or LPS-PBMCs (Fig. 5H; p = 0.35; repeated measures one-way ANOVA). In hippocampal neurons, OCHSCs co-cultured with IR-PBMCs and LPS-PBMCs showed significantly lower ID than naive OCHSCs (Fig. 5J, K and N; IR-PBMCs: p < 0.0001; LPS-PBMCs: p < 0.0001; repeated measures one-way ANOVA) as well as control PBMC-OCHSC co-cultures (Fig. 5N; IR-PBMCs: p < 0.0001; LPS-PBMCs: p < 0.001; repeated measures one-way ANOVA), whereas adding control PBMCs did not result in significant changes in ID compared to naive OCHSCs (Fig. 5N; p = 0.06; repeated measures one-way ANOVA).
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.
4.5. 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 inflammation transduction solely by binding to their cognate receptors found in OCHSC cells (29). 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, Fig. 2), we added PBMCs to OCHSC media to allow PBMC-derived cytokines and chemokines, but not PBMCs themselves, to diffuse through the membrane. In the next set of experiments, we focused on the effects of media-incubated PBMCs on inflammasome assembly and neuronal excitability in the hippocampus.
Immunohistochemical analysis of the inflammasome markers NLRP1, NLPR3 and ASC in OCHSCs revealed that media incubation of IR-PBMCs (mIR-PBMCs) gave rise to a stark upregulation of NLRP1 (Fig. 6G and J; p < 0.0001; one-way ANOVA), NLRP3 (Fig. 6H and K; p < 0.01) and ASC (Fig. 6I, I´ and L; p < 0.001; one-way ANOVA) in the hippocampus. On the other hand, while NLRP1 was overexpressed following media incubation of LPS-PBMCs (mLPS-PBMCs) (Fig. 6D and J; p < 0.05; one-way ANOVA), there was a non-significant trend of NLRP3 upregulation (Fig. 6E and K; p = 0.07; one-way ANOVA) and no detectable expression of ASC (Fig. 6F, F´ and L; p > 0.99; one-way ANOVA) as compared to media incubation of control PBMCs (mCTL-PBMCs) (Fig. 6A-C´ and J-L). In addition, the expression level of NLRP1 and ASC, but not NLRP3, was significantly higher when OCHSCs were incubated with mIR-PBMCs than with mLPS-PBMCs (Fig. 5J, K and L; NLRP1: p < 0.05, NLRP3: p = 0.42, ASC: p < 0.01; one-way ANOVA). The presence of NLRP1/3 inflammasomes scaffolding protein ASC in the mIR-PBMCs group supports the hypothesis that IR-PBMCs can induce inflammasome oligomerization in OCHSCs through diffusible mediators, whereas the failure of mLPS-PBMCs to evoke similar effect suggests that LPS-PBMCs require cell-cell contact with neural tissue for inflammasome activation.
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 (Fig. 5P-R; Rin: p = 0.41, rheobase: p = 0.93, APR: p > 0.99; one-way ANOVA), and consistent with the absence of hippocampal inflammasome activation in OCHSCs following mLPS-PBMCs. However, in hippocampal neurons exposed to mIR-PBMCs, Rin, rheobase and APR values were significantly different from mCTL-PBMCs (Fig. 5M-R; Rin: p < 0.01, rheobase: p < 0.05, APR: p < 0.05; one-way ANOVA), again consistent with inflammasome induction in OCHSCs from this group. Hence, in contrast to adding LPS-PBMCs on top of OCHSCs (Sect. 4.3, Fig. 4S-U), the outcome of mLPS-PBMCs was comparable to that of mCTL-PBMCs, indicating that media application of LPS-PBMCs was sufficient to enhance neuronal excitability. Also, these results demonstrate that similar to IR-PBMCs, mIR-PBMCs retained their capacity to enhance neuronal excitability.
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 (Fig. 5T and V; IA: p > 0.99, ID: p > 0.99; repeated measures one-way ANOVA). 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 (Fig. 5S-V; IA: p < 0.01, ID: p < 0.05; repeated measures one-way ANOVA) and mLPS-PBMCs (Fig. 5T and V; IA: p < 0.01, ID: p < 0.01; repeated measures one-way ANOVA). The fact that a 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 summary, with respect to the hippocampus, mIR-PBMCs mirrored the effects of co-culturing IR-PBMCs with OCHSCs, reproducing inflammasome formation and ID-/IA-mediated hyperexcitability, thereby suggesting that diffusion of inflammatory factors secreted by mIR-PBMCs, likely cytokines, rather than cell-cell contact is responsible of these phenotypic changes observed in OCHSCs. On the contrary, mLPS-PBMCs unexpectedly failed to provoke inflammasome assembly or neuronal hyperexcitability; an outcome that was distinct from LPS-PBMC-OCHSC co-cultures. Thus, in the case of LPS-PBMCs, cell-cell contact seems to be essential for transducing peripheral inflammatory signals from LPS-PBMCs to the hippocampus in OCHSCs.
4.6. Neuroinflammation-mediated attenuation of 4-AP sensitive transient K + currents is dependent on Caspase-1 activation in hippocampal cells
Pharmacological inhibition of Caspase-1 cleavage using the selective inhibitor VX-765 has proved to be highly effective in reducing inflammasome activation in the CNS (39–41). To test if Caspase-1 inhibition could mitigate PBMC-induced neuroinflammation in the hippocampus, OCHSCs were pre-treated with VX-765 one hour before seeding LPS-PBMCs (VX765-LPS-PBMCs) and IR-PBMCs (VX765-IR-PBMCs) on top of OCHSCs. Thereafter, we analyzed the expression profile of inflammasome markers and measured neuronal excitability following the co-culturing of 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 control group.
We found notable augmentation of NLRP1, NLRP3 and ASC immunolabeling in the DMSO-LPS-PBMCs group (Fig. 7A-C´ and J-L) as opposed to the VX765-LPS-PBMCs (Fig. 7D-F´ and J-L; NLRP1: p < 0.01, NLRP3: p < 0.01, ASC: p < 0.001; one-way ANOVA) and the VX765-IR-PBMCs groups (Fig. 7G-I´ and J-L; NLRP1: p < 0.01, NLRP3: p < 0.001, ASC: p < 0.001; one-way ANOVA). Thus, VX-765 is a potent inhibitor of PBMC-transduced inflammatory signals 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-PBMC (Fig. 7M-R; Rin: p < 0.01, rheobase: p < 0.05, APR: p < 0.05; one-way ANOVA) and VX765-IR-PBMC co-cultures with OCHSCs (Fig. 7M-R; Rin: p < 0.05, rheobase: p < 0.05, APR: p < 0.05; one-way ANOVA). Further, IA and ID amplitudes of hippocampal neurons from DMSO-LPS-PBMC co-cultures were notably reduced versus VX765-LPS-PBMC (Fig. 7S-V; IA: p < 0.001, ID: p < 0.001; repeated measures one-way ANOVA) and VX765-IR-PBMC co-cultures with OCHSCs (Fig. 7S-V; IA: p < 0.01, ID: p < 0.05; repeated measures one-way ANOVA).
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 an underlying mechanism of PBMC-induced IA- and ID-mediated enhanced excitability.