DLL4 is expressed by CNS astrocytes and upregulated during chronic neuroinflammation.
First, we showed that DLL4 is strongly upregulated in reactive astrocytes under inflammatory conditions in vitro using CNS normal human astrocytes (NA) from ScienCell treated with IL-1β (interleukin-1β), a central pro-inflammatory cytokine driving multiple sclerosis pathophysiology (Fig. 1, A, C-D) and in vivo (Fig. 1, E), in the neurovascular unit, using experimental autoimmune encephalomyelitis (EAE), a pre-clinical model of multiple sclerosis using MOG35 − 55 to induce chronic neuroinflammation in C57BL/6 mice. For this experiment, isolated spinal cord micro-vessels underwent a digestion step followed by a CD45+ T cell depletion step to eliminate inflammatory cell infiltrates induced by EAE. DLL4 upregulation in human reactive astrocytes in vitro is associated with upregulation of the astrocyte reactivity marker VIM (Vimentin) (Fig. 1, B). Dll4 upregulation in the neurovascular unit is associated with upregulation of Notch signaling activation markers Hey1 (hairy/enhancer-of-split related with YRPW motif protein 1) and Jag1 (jagged 1) (Fig. 1, F-G). We then confirmed that DLL4 is upregulated in reactive astrocytes in vivo, on spinal cord sections from EAE induced C57BL/6 adult mice (Fig. 1, H-I) and on cortical active lesions from multiple sclerosis patients (Fig. 1, J). In addition, we measured astrocytic DLL4 expression level in cervical, thoracic and lumbar spinal cord sections from EAE induced C57BL/6 adult mice at different time points (day 0, 5, 11, 18 and 24 post immunization). We showed that astrocytic expression of DLL4 is predominant at the thoracic level of the spinal cord in the early stage of EAE (day 5 post immunization), before increasing at the cervical level of the spinal cord in the late stages of EAE (day 11 and 18 post immunization). DLL4 remains weakly expressed by astrocytes at the lumbar level of the spinal cord regardless of the post immunization time point (Fig. 1, K).
Inactivation of astrocyte Dll4 reduces disability in a model of multiple sclerosis during the onset and plateau of the disease.
To test the role of astrocyte DLL4 in neuroinflammation, we conditionally disrupted Dll4 expression in astrocytes using 2 different promoters (the Glast-CreERT2 promoter and the Aldh1L1-CreERT2 promoter) and examined the consequences on EAE pathology. Experimental mice consisted of Glast-CreERT2, Dll4Flox/Flox mice or Aldh1L1-CreERT2, Dll4Flox/Flox mice with corresponding littermate controls (Dll4Flox/Flox). We first verified the efficiency of both knockouts, 4 months after inducing knockdown by intra-peritoneal injection of tamoxifen. The Glast-CreERT2 promoter induced a moderate recombination (30–50%) whereas the Aldh1L1-CreERT2 promoter induced a full recombination (90–100%) in both spinal cord and cortical astrocytes (Supplemental Fig. 1, A-H). The difference in recombination efficiency enabled us to test the effects of partial versus complete astrocyte Dll4 knockdown in the disease course and spinal cord pathology of experimental multiple sclerosis (EAE).
In the rest of the manuscript, Glast-CreERT2, Dll4Flox/Flox mice will be named Dll4ACKOP (for partial recombination) and Aldh1L1-CreERT2, Dll4Flox/Flox mice will be named Dll4ACKOC (for complete recombination) to make reading easier.
We confirmed the absence of DLL4 astrocyte expression in non-reactive astrocytes in Dll4ACKOC mice versus control littermates (Supplemental Fig. 1, I) and highlighted astrocyte specific DLL4 downregulation in EAE induced Dll4ACKOC mice versus control littermates (Supplemental Fig. 1, J) and in Dll4ACKOP mice versus control littermates injected in the cortex with AdIL-1β (Supplemental Fig. 1, K)), another mouse model of astrocyte reactivity and CNS autoinflammation.
In the Dll4ACKOP mouse model, littermate control mice demonstrated neurologic deficits from day 11, which increased in severity until day 21, when the clinical score stabilized at a mean of 3.0, representing hind limb paralysis. In contrast, the clinical course in Dll4ACKOP mice was much milder: disease reached a plateau at day 27 at a mean of 2.5, indicating hind limb weakness and unsteady gait, a milder phenotype (Fig. 2, A). The EAE peak score (Fig. 2, B) was no different between the groups but average score during the time of disability (Fig. 2, C) was decreased in Dll4ACKOP mice.
In the Dll4ACKOC mouse model, littermate control mice exhibited neurologic deficits from day 12, which increased in severity until day 20, when clinical score stabilized at a mean of 2.8, representing hind limb paralysis. In contrast, the onset of clinical signs in Dll4ACKOC mice was first seen four days later, and the clinical course was very mild. Indeed, in Dll4ACKOC mice, disease reached a plateau at day 18 at a mean of 0.4 indicating almost no sign of paralysis (Fig. 2, D). Strikingly, only two Dll4ACKOC mice developed very mild symptoms, the remaining majority showing no sign of pathology other the course of the disease. The EAE peak (Fig. 2, E) and average scores during the time of disability (Fig. 2, F) were both strongly decreased in Dll4ACKOC mice.
Clinical course in both Dll4ACKOP mice and Dll4ACKOC mice was correlated with decreased areas of demyelination as compared to the control cohorts (Fig. 2, G-J).
In sum, we found that clinical course and pathology of EAE are reduced in mice with astrocyte Dll4 knockdown. Furthermore, degree of Dll4 knockdown appeared to have a dose-dependent effect as reduced pathology was only observed during the onset of the disease in the Dll4ACKOP mice while reduced pathology was observed during both the onset and plateau of the disease in the Dll4ACKOC mice. Moreover, the magnitude of the protective effect of Dll4 astrocyte knockdown was stronger in Dll4ACKOC mice than in Dll4ACKOP mice.
Astrocyte-specific Dll4 inactivation induces downregulation of astrocyte reactivity under neuroinflammatory condition both in vitro and in vivo.
To test the importance of astrocyte DLL4 expression in reactive astrocytes during neuroinflammation, we conditionally disrupted DLL4 expression in human astrocytes, and examined the consequences on astrocyte reactivity. To do so, we used human Normal Astrocytes (NA) from ScienCell that we transfected with either a siRNA control or a siRNA targeting Dll4 expression. Astrocyte reactivity was then induced using the pro-inflammatory cytokine Il-1β to induce astrocyte reactivity in vitro.
We first verified the efficiency of the knockdown by measuring DLL4 gene and protein expression in primary human NA cultures transfected with the siCTRL versus siDLL4 and treated with IL-1β, and showed that DLL4 gene and protein expression were strongly downregulated in the siDLL4 condition compared to the siCTRL condition (Fig. 3, A-C) along with the protein expression of NICD1 (notch1 intracellular domain) which reflects the level of activation of the notch pathway (Fig. 3, D-E). The downregulation of the DLL4-NOTCH1 axis was paired with the downregulation of identified reactive astrocyte markers notably VIM (VIMENTIN) (28) and cleaved CASPASE 3 (29) (Fig. 3, D, F-G). We then confirmed that Dll4 knockdown leads to disruption of astrocyte reactivity in vivo, on isolated astrocyte lysates from EAE induced Dll4ACKOP and Dll4ACKOC mice and control littermates.
Transcriptional profiling of isolated astrocyte lysates from EAE induced Dll4ACKOC and control littermates showed that 1558 genes were downregulated in EAE induced Dll4ACKOC mice while 874 genes were upregulated (Supplemental Fig. 2, A). Notably, among the downregulated genes, a wide cohort of transcripts linked to reactive astrocyte markers (Fig. 3, H). Specifically, this approach identified, among others, Vim (vimentin) and Serpina3n transcripts as downregulated in astrocyte samples from EAE induced Dll4ACKOC mice (Fig. 3, H). Importantly, these two factors, like all the genes highlighted in the heatmap (Fig. 3, H), have been identified as markers of astrocytic reactivity in the international consensus published in 2021 in the journal nature neuroscience(28). The pro-inflammatory cytokine Il-6 (interleukin-6) transcripts are also downregulated in astrocyte samples from EAE induced Dll4ACKOC mice (Fig. 3, H). Surprisingly, Gfap transcripts weren’t modulated in astrocyte samples from EAE induced Dll4ACKOC mice (cf transcriptional profiling of isolated astrocyte lysate full table, additional file 1). However, in examining VIM and GFAP protein expression by western blot, in spinal cord lysates from Freund adjuvant and EAE induced Dll4ACKOC mice compared to control littermates; we showed that both factors are downregulated in Dll4ACKOC mice (Fig. 3I-K). Moreover, in examining VIM and GFAP protein expression by immunofluorescence on spinal cord sections from EAE-induced Dll4ACKOP mice and control littermates and from EAE-induced Dll4ACKOC mice and control littermates, we confirmed that both GFAP and VIM expression are decreased in the CNS of astrocyte specific Dll4 deficient mice (Dll4ACKO mice) (Fig. 3, L-N) (Supplemental Fig. 2, B).
These findings indicate astrocytic DLL4 expression contributes to the induction of astrocyte reactivity under neuroinflammatory conditions.
Astrocyte-specific Dll4 activity depends partially on its interaction with astrocytic Notch1 receptor in vivo and drives NICD upregulation in astrocytes leading to the upregulation of IL-6 transcripts via a direct interaction with NICD.
Next, we explored the signaling factors downstream of DLL4 induced astrocyte reactivity. To test if DLL4 induced astrocyte reactivity depends on its interaction with NOTCH1 receptor in reactive astrocytes; we conditionally disrupted Notch1 expression in astrocytes using the Aldh1L1-CreERT2 promoter and examined the consequences on EAE pathology. Experimental mice consisted of Aldh1L1-CreERT2, Notch1Flox/Flox mice with corresponding littermate controls (Notch1Flox/Flox). In the rest of the manuscript, Aldh1L1-CreERT2, Notch1Flox/Flox mice will be named Notch1ACKOC to make reading easier.
In the Notch1ACKOC mouse model, control mice exhibited neurologic deficits from day 12, which increased in severity until day 22, when clinical score stabilized at a mean of 2.8, representing hind limb paralysis. In contrast, the clinical course in Notch1ACKOC mice was much milder: disease reached a plateau at day 20 at a mean of 1.7, indicating hind limb weakness and unsteady gait, a milder phenotype (Fig. 4, A). These results confirm that astrocytic knockouts of Dll4 or Notch1 both lead to a decrease in EAE disease progression with recovery of hind limb mobility in knockout animals compared to littermate controls. Thus, these data strongly support our hypothesis that DLL4 is indeed acting through NOTCH1 on reactive astrocytes during neuro-inflammation (Fig. 4, A).
Interestingly, the impact of astrocytic Notch1 knockout (Fig. 4, A) is more moderate than that of astrocytic Dll4 knockout (Fig. 2, D) in the context of EAE. This means that astrocytic DLL4 interacts with its NOTCH1 receptor in activated astrocytes, but also potentially with other neighboring cells, via the DLL4-NOTCH signaling, to impact EAE pathology. Given the structure of the neurovascular unit and the inflammatory context associated with EAE, we assume that these neighboring cells might be the lymphocytes infiltrating the CNS parenchyma. This hypothesis remains to be explored and is discussed in the discussion.
Interestingly, NICD has been found to directly regulate IL-6 (interleukin-6) expression in activated macrophages (30) and our transcriptional RNA profiling of astrocyte lysates from EAE induced Dll4ACKOC and control littermates revealed a strong downregulation of Il-6 transcripts in Dll4ACKOC samples compared to controls (Fig. 3, H). Therefore, we hypothesized that a direct interaction between NICD and IL-6, already observed in reactive macrophages, may contribute to reactive astrocyte reactivity pathways in vitro. To respond to this question, we performed a ChIP experiment on reactive astrocyte lysates using IgG versus NICD antibodies to do the pull down and human IL-6 primers for the PCR and demonstrated that NICD directly interacts with IL-6 gene promoter in reactive astrocytes in vitro (Fig. 4, B). Then, to rule out any potential effect of the inflammatory factor IL-1β on the upregulation of IL-6 in our in vitro model, we transduced non-reactive human astrocytes with a DLL4 expressing lentivirus versus an empty lentivirus. First we validated that the DLL4-NOTCH1 pathway was activated in the astrocyte cultures transduced with the DLL4 expressing lentivirus, showing the upregulation of HES1, HEY1 and HEY2 genes (Fig. 4, C-E) and highlighted the concurrent upregulation of IL-6 expression level (Fig. 4, F). DLL4 upregulation was also validated to ensure the transduction efficacy (Fig. 4, G).
Altogether, these results suggest that DLL4 driven NOTCH1 activation in reactive astrocytes leads to the upregulation of IL-6 transcripts via a direct interaction between NICD and the gene coding for IL-6.
Astrocyte-specific Dll4 inactivation induces downregulation of astrocyte reactivity through the downregulation of the IL-6-STAT3 pathway both in vitro and in vivo.
JAK/STAT (janus kinase/signal transducer and activator of transcription) signaling is an essential effector pathway for the development and regulation of immune responses. Unbridled activation of the JAK/STAT pathway by pro-inflammatory cytokines, notably IL-6, plays a critical role in driving the pathogenesis of multiple sclerosis/EAE (31). Moreover, STAT3 has been shown to control astrocyte reactivity in various pathologies such as ischemic stroke, neuroinflammatory disorders and brain tumors (32–34). We tested whether the IL-6-JAK/STAT pathway was regulated by astrocytic Dll4 in vitro and in vivo. First we showed that both IL-6 and P-STAT3 (phosphorylated form of STAT3) were downregulated in reactive human astrocytes transfected with the DLL4 siRNA compared to reactive human astrocytes transfected with the CTRL siRNA. Notably, IL-6 and P-STAT3 expression level in the DLL4 siRNA condition was similar to the one measured in non-reactive astrocytes (Fig. 5, A-C). Under the same conditions in vitro and on spinal cord sections from Dll4ACKOC mice and control littermates, we then demonstrated that IL-6 signal was co-localized with GFAP signal in reactive astrocytes (Fig. 5, D-E) (Supplemental Fig. 2, C) and that IL-6 expression level was downregulated following Dll4 knockdown both in vitro and in vivo (Fig. 5, D-G) (Supplemental Fig. 2, C). To clearly establish the link between IL-6 and P-STAT3, we then measured the phosphorylation of STAT3 in vitro, in human reactive astrocytes treated with Tocilizumab, a humanized monoclonal antibody targeting IL-6 receptors, or IgG. We showed that P-STAT3 is downregulated after Tocilizumab treatment compared to IgG with an expression level similar to the one of non-reactive astrocytes (Fig. 5, H-I). Importantly, DLL4 expression level was stable in both IgG and Tocilizumab treated reactive astrocytes, highlighting that IL-6-STAT3 interaction happened downstream of DLL4 during astrocyte reactivity (Fig. 5, H, J).
Here we showed that, following the direct upregulation of IL-6 transcription by NICD in reactive astrocytes, IL-6 protein expression is strongly increased and leads to the phosphorylation of STAT3, an already established marker of astrocyte reactivity via its interaction with the kinase JAK.
Mice with astrocyte Dll4 inactivation display resistance to increases in blood-brain barrier permeability is associated with decreases in VEGFA and TYMP secretion, protecting the parenchyma from inflammatory infiltrate in a model of multiple sclerosis and in a model of acute neuroinflammation.
We compared the impact of Dll4 blockade in astrocytes on plasma protein and inflammatory cell infiltration in the parenchyma by measuring IgG, CD4 and CD45 + lymphocyte infiltration and IBA1 expression level in vivo, on spinal cord sections from EAE induced Dll4ACKOP and Dll4ACKOC mice versus control littermates (Fig. 6, A-D) (Supplemental Fig. 3, A-H). We found that astrocyte Dll4 deficient mice induced with EAE displayed less parenchymal inflammatory infiltration than control littermates (Fig. 6, A-D) (Supplemental Fig. 3, A-H). We previously reported that reactive astrocytes express pro-permeability factors, VEGFA and TYMP, which drive blood-brain barrier permeability in EAE (7). We therefore tested whether astrocyte DLL4 signaling regulates VEGFA and TYMP expression in vitro in DLL4 siRNA treated reactive astrocytes and in vivo in EAE induced Dll4ACKOP and Dll4ACKOC mice. We found that VEGFA and TYMP were downregulated in IL-1β induced reactive astrocytes transfected with DLL4 siRNA when compared to IL-1β induced reactive astrocytes transfected with CTRL siRNA (Fig. 6, E-H). Moreover we showed that TYMP expression level was highly correlated to astrocyte reactivity threshold as it followed the same pattern as GFAP signal intensity (Fig. 6, H, J-K). We then confirmed these results in vivo, finding downregulation of astrocyte VEGFA and TYMP signals on spinal cord sections from EAE induced Dll4ACKOP and Dll4ACKOC mice when compared to control littermates (Fig. 6, I, L-M) (Supplemental Fig. 3, I-K).
Altogether, these results suggest that, under neuroinflammatory condition, astrocyte Dll4 knockdown leads to decreased astrocyte reactivity and is associated with reduced levels of VEGFA and TYMP, protecting against blood-brain barrier breakdown and parenchymal inflammatory infiltrate.
To further support whether astrocyte DLL4 participates to inflammatory lesion pathogenesis by controlling astrocyte reactivity, we decided to verify the results we obtained in the EAE mouse model, in a model of acute neuroinflammatory lesion. Initially, we compared responses of Dll4ACKOP mice and littermate controls to cortical injection of AdIL-1β, measuring the area of neuronal cell death (NEUN loss) in lesions at 7 dpi (days post injection) (Supplemental Fig. 4, A, F). We then measured astrocyte reactivity (VIM and GFAP signal intensity) and associated IL-6 and TYMP expression in lesions at 7 dpi (Supplemental Fig. 4, B-C, G-J). Finally, the maximal area of inflammatory infiltration into the CNS in term of parenchymal entry of serum proteins, notably FGB (fibrinogen), and CD4+ T helper lymphocytes was assessed in lesions at 7 dpi (Supplemental Fig. 4, D-E, K-L). Importantly, these studies demonstrated that lesion formation in Dll4ACKOP mice was strongly decreased compared to littermate controls. Confirming efficacy and specificity of inactivation, AdIL-1β–induced lesions in Dll4ACKOP mice showed lower levels of DLL4 (Supplemental Fig. 1, K). Lesion size in Dll4ACKOP mice, as measured by neuronal cell death or loss of NEUN immunoreactivity, was much narrower than in controls at 7 dpi (Supplemental Fig. 4, A, F). Moreover, GFAP and VIM, two markers of astrocyte reactivity were strongly downregulated in Dll4ACKOP mice compared to controls and were associated with a decreased expression of astrocyte reactivity marker IL-6 and pro-permeability marker TYMP (Supplemental Fig. 4, B-C, G-J). There were also large decreases in the areas of cortical FGB and CD4+ T helper lymphocytes infiltration seen in AdIL-1β lesions in Dll4ACKOP mice at 7 dpi (Supplemental Fig. 4, D-E, K-L).
We concluded that astrocyte DLL4 upregulation during acute neuroinflammation leads to astrocyte reactivity in the lesion area and upregulation of the pro-inflammatory cytokine IL-6 and pro-permeability factor TYMP by reactive astrocytes. Lesion size is more severe in presence of astrocytic DLL4 and associated with a stronger inflammatory infiltrate of fibrinogen and immune cells into the parenchyma. Altogether our findings in both cortical AdIL-1β lesions and spinal cord EAE support a global role for astrocytic Dll4 in driving astrocyte reactivity and neuroinflammatory lesion pathogenesis.
Blockade of DLL4 protects against blood-brain barrier opening and paralysis in EAE
To test the therapeutic potential of exogenous DLL4 blockade during EAE, we designed experiments to test the effects of an anti-DLL4 antibody. To probe for possible off-target effects of Dll4 blockade on the vascular endothelium, we first tested the EAE phenotype in a transgenic mouse with conditional endothelial Dll4 inactivation as DLL4 contributes to the regulation of angiogenesis via DLL4-mediated NOTCH1 signaling in endothelium, a key pathway for vascular development (35). Therefore, we induced EAE in 10 weeks old Cadherin5-CreERT2, Dll4Flox/Flox mice and corresponding littermate controls and found that Dll4 endothelial specific downregulation had no impact on EAE disease severity (Supplemental Fig. 5, A), associated peak (Supplemental Fig. 5, B) and average score during time of disability (Supplemental Fig. 5, C). Moreover, in C57BL/6 spinal cord EAE lesions, we showed that DLL4 is not expressed by SMA + mural cells (Supplemental Fig. 5, D-E). Altogether, these data suggest that exogenous Dll4 blockade with an anti-Dll4 antibody would have minimal effects on the vascular endothelium impacting the course or neuropathology of EAE.
We then compared the impact of DLL4 blockade on disease severity in EAE (Fig. 7). We sensitized 8 weeks old C57BL/6 mice (9 per group) and, beginning on day 8 post sensitization (just at the beginning of the onset of the disease), treated them every three days for eleven days with the inVivoMab anti-mouse DLL4 antibody (500 µg/mouse/d). The inVivoMab polyclonal Armenian hamster IgG (500 µg/mouse/d) was injected to the mice in the control group. Neurological signs and pathology were evaluated from day 1 to day 25 post sensitization.
Onset of the disease occurred at day 10 post-sensitization, and ascending paralysis was first observed 24 h later, with severity in controls (polyclonal Armenian hamster IgG-treated mice) increasing until day 19, when neurologic deficit reached a plateau at a mean score of 3.3, indicating severe disease with complete hindlimb paralysis (Fig. 7, A). Signs in anti-mouse DLL4 antibody-treated mice were much milder, peaking at day 16 at a mean score of just 2.4, indicative of a limp tail with moderate hindlimb weakness (Fig. 7, A). Comparison of peak clinical severity in each individual was different between the anti-mouse DLL4 antibody and the polyclonal Armenian hamster IgG-treated regimens (Fig. 7, B). For anti-mouse DLL4 antibody, differences in disease severity were highly significant compared with polyclonal Armenian hamster IgG-treated controls (Fig. 7, C). Almost all controls (89%) but only 33% of anti-mouse DLL4 antibody-treated mice displayed complete hindlimb paralysis or worse (score ≥ 3) (Fig. 7, A-C). No mortality at all was encountered in all cohorts.
These improvements in clinical disease observed with the anti-mouse DLL4 antibody were associated with reduced tissue damage, in terms of decreased demyelination (Fig. 7, D, H). They were also associated with reduced blood–brain barrier breakdown, plasmatic protein and inflammatory cell infiltration (Fig. 7, E-F, J-L) and astrocyte reactivity (Fig. 7, G-H, M-N). No difference was observed in term of microglial activation (Fig. 7, D, K). Downregulation of astrocyte reactivity in the anti-mouse DLL4 antibody group was associated to a decreased expression of the pro-inflammatory cytokine IL-6 and of the pro-permeability factors TYMP and VEGFA (Fig. 7, G-H, O-P).
Collectively, the results of these therapies confirm the therapeutic benefit of exogenous DLL4 blockade during EAE and support our findings that astrocyte DLL4 promotes astrocyte reactivity during neuroinflammation, which contributes to TYMP and VEGFA astrocytic secretion leading to blood-brain barrier permeability.