Background
Pathophysiology of spinal cord injury (SCI) is characterized by a robust humoral and cellular neuroinflammatory response that is driven by the interplay between the peripherally recruited leukocytes and resident glial cells [
1,
2]. Microglia/macrophages, T cells, and B cells play central roles in orchestrating the innate and adaptive immune responses following SCI through a plethora of inflammatory cytokines, chemokines, antibodies, and proteolytic enzymes [
2‐
4]. Activated microglia/macrophages mediate the initial neuronal injury through pro-inflammatory mediators and oxidative damage [
5]. They also activate T cells through their antigen presenting function [
1]. Activated T cells stimulate B cells, through a host of cytokines and signaling molecules, to produce autoreactive antibodies against spinal cord tissue [
6] causing tissue damage through antibody-mediated cytotoxicity [
7,
8]. These processes collectively lead to an imbalanced and dysregulated milieu that impedes repair and regeneration after SCI [
1]. Despite their undisputed role in degenerative processes following SCI, emerging evidence indicates that immune cells can be modulated in their microenvironment to adopt regulatory and pro-regenerative phenotypes [
9,
10]. Given the profound impact of neuroinflammation on SCI pathophysiology, it is critical to unravel endogenous mechanisms that regulate immune cells after injury. This knowledge is vital for developing immunotherapies that can harness the potential of immune cells in fostering a supportive microenvironment for spinal cord repair and regeneration.
We previously identified that SCI results in a rapid and long-lasting decline in the tissue levels of the neuronally derived growth factor, Neuregulin-1 (Nrg-1) [
11]. Nrg-1 is primarily known for its essential role in Schwann cell and oligodendrocyte differentiation, maintenance, and myelination in the central and peripheral nervous systems [
12]. In a preclinical model of compressive/contusive SCI in rats, we demonstrated that increasing the deficient bioavailability of Nrg-1 in the injured spinal cord improves neurological recovery following injury [
13]. In our efforts to elucidate the mechanisms underpinning the recovery of function after Nrg-1 treatment [
13], we identified that Nrg-1 promotes oligodendrogenesis and protects oligodendrocytes and axons resulting in white matter preservation after SCI [
11]. Importantly, we found a remarkable positive role for Nrg-1 in regulating astrogliosis and glial scar formation in the injured spinal cord [
13]. Moreover, Nrg-1 treatment through intrathecal infusion attenuated the release of pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-⍺), and interleukin-1 beta (IL-1β) in acute SCI while increasing the tissue levels of the anti-inflammatory cytokine, IL-10 at subacute SCI [
13]. Other studies have also shown a neuroprotective role for Nrg-1 in attenuating neuronal injury in vitro and in ischemic brain injury models by reducing neurotoxicity and pro-inflammatory mediators [
14,
15]. These initial findings suggest a potential immunomodulatory mechanism of Nrg-1 in fostering a pro-regenerative microenvironment that improves tissue preservation and neurological recovery following SCI [
13].
In the present study, we dissected the impact of Nrg-1 on the peripheral and spinal cord immune responses at various stages of SCI. Using a clinically relevant model of severe compressive SCI in rat, we investigated whether systemic delivery of recombinant human Nrg-1 (rhNrg-1) could regulate the recruitment, phenotype, and secretory properties of SCI relevant leukocytes in the blood and injured spinal cord. We demonstrate, for the first time, that Nrg-1 promotes a comprehensive immune regulatory response by macrophages and T and B lymphocytes at acute and chronic stages of SCI through modulation of a repertoire of cytokines and chemokines in the spinal cord tissue. Our new findings establish that bioavailability of Nrg-1 activates a neuroinflammatory process that provides a supportive environment for endogenous repair and recovery of function following SCI.
Discussion
In the current study, using a clinically relevant model of compressive/contusive SCI in rats, we have identified a novel positive immunomodulatory role for Nrg-1 in SCI. Systemic Nrg-1 therapy augments regulatory populations of macrophages, T cells, and B cells both peripherally and in the injured spinal cord tissue during the acute and chronic phases of SCI. Moreover, Nrg-1 treatment promotes pro-regenerative immune mediators such as IL-10 and CCL11 while attenuating pro-inflammatory cytokines and chemokines, IL-6, IFN-γ, CXCL1, CXCL2, and CXCL3. Importantly, Nrg-1 positively regulates B cell activity and moderates SCI-induced deposition of IgG and IgM in the spinal cord. To our knowledge, this is the first study that investigates the impact of Nrg-1 on neuroinflammation in traumatic SCI. Importantly, we have demonstrated the potential of systemic Nrg-1 as a new promising immunomodulatory strategy for traumatic SCI in a clinically relevant model. Of note, the Nrg-1 treatment used in our study is shown to pass the blood-brain and spinal cord barrier and enter the central nervous system (CNS) readily [
46] and importantly has shown therapeutic efficacy in previous studies by our group and others [
13,
47].
Nrg-1 and ErbB network is known for its critical role in the developing central and peripheral nervous systems [
48]. In the spinal cord, while the implication of Nrg-1 in neural differentiation and myelination is established during development [
49,
50], our knowledge on the role of Nrg-1 in SCI pathophysiology and neuroinflammation is still in its infancy. In recent years, work by our group and others has begun unraveling the importance of Nrg-1 in the pathologic CNS [
11,
13‐
15]. We originally identified that Nrg-1 protein expression is severely depleted in acute traumatic SCI and remains down-regulated chronically [
11]. We established a close correlation between Nrg-1 dysregulation and impaired endogenous precursor response after SCI [
11]. Restoration of Nrg-1 was sufficient to activate an endogenous repair program that promoted oligodendrocyte replacement and white matter repair following SCI [
11,
13]. A recent study has also shown an essential role for Nrg-1 in Schwann cell driven remyelination after SCI that is known to be instrumental for endogenous myelin repair in the injured spinal cord [
12]. Interestingly, we found that Nrg-1 treatment exerts a prominent neuroprotective effect on oligodendrocytes and axons after SCI [
11], which complements the recent reports showing increased neuronal survival following ischemic brain insult [
15]. In addition to the anticipated impact of Nrg-1 on oligodendrocytes and myelination, our recent in vitro and in vivo studies uncovered a novel role for Nrg-1 in regulating astrocyte response to injury and evolution of the glial scar in the injured spinal cord [
13]. More specifically, Nrg-1 treatment had a remarkable effect on matrix remodeling in the glial scar by moderating the SCI-induced upregulation of chondroitin sulfate proteoglycans (CSPGs), a key regulator of SCI pathophysiology and a master inhibitor of repair processes [
51]. We unraveled that Nrg-1 mediates its effects through activation of ErbB2/3 complex in glial cells and an increase in phosphorylation of Erk1/2 and STAT3 pathways following SCI [
13]. Most importantly, we showed that Nrg-1 treatment improves neurobehavioral recovery in a dose-dependent manner after SCI [
13]. Interestingly, these studies also provided the initial evidence for immunomodulatory effects of Nrg-1, which seems to be an underpinning mechanism for its beneficial effects in SCI.
We and others have demonstrated that Nrg-1 positively influences microglia in vitro and attenuates their response to stressful conditions as evidenced by the reduced production of pro-inflammatory mediators such as nitric oxide (NO), IL-1β, and TNF-⍺ [
13,
52]. In rat SCI, we found that intrathecal Nrg-1 treatment can remarkably enhance an IL-10 dominant cytokine balance [
13] that is shown to be beneficial for SCI repair processes such as remyelination [
53]. Several immune regulatory populations such as T
reg cells, B
reg cells, and M2 microglia/macrophages produce IL-10 after SCI suggestive of a broader role for Nrg-1 in modulating neuroinflammation [
54‐
56]. Notably, T and B lymphocytes, macrophages, and microglia express Nrg-1 receptors [
13,
57‐
59], and therefore, all these populations can potentially be affected by the dysregulation of Nrg-1 signaling following SCI. To date, the role of Nrg-1 in modulating the innate and adaptive immune response in SCI has remained elusive. In this study, we employed systemic Nrg-1 delivery to understand how Nrg-1 influences the recruitment and function of immune cells not only within the injured spinal cord tissue but also in the peripheral blood. Additionally, systemic delivery provides a more clinically relevant therapeutic strategy for SCI.
Our immunophenotypic investigation of the injured spinal cord tissue revealed that systemic Nrg-1 increases macrophage infiltration into the injured spinal cord, which seems to be temporally regulated. We found that Nrg-1 promotes the recruitment of macrophages acutely and chronically with no apparent effect at the subacute stage. Interestingly, previous studies on acute peripheral nerve injury have also shown that intrathecal infusion of Nrg-1 induces recruitment and proliferation of resident microglia in the dorsal horn where injured sensory afferents enter the spinal cord [
60,
61]. Increased microgliosis and macrophage proliferation in response to over-activation of Nrg-1 and ErbB receptors have been also shown in direct in vitro studies [
60,
61]. Although Nrg-1-induced microgliosis has been associated with neuropathic pain in peripheral nerve injury [
60,
61], our previous studies in rat SCI showed no significant change in pain sensation following Nrg-1 treatment [
13]. It is noteworthy that Nrg-1 treatment in our SCI studies augments the downregulated levels of Nrg-1 in the injured spinal cord as opposed to over-activating its signaling. Interestingly, although Nrg-1 increased the population of macrophages in acute SCI, this increase mainly reflected an elevation in the subpopulation of alternatively activated M2 macrophages (CD45
+CD68
+CD163
+IL10
+) with no change in the population of classically activated pro-inflammatory M1 (CD45
+CD68
+CD86
+) macrophages. Notably, the increase in M2 macrophage population at the acute stage of SCI is in agreement with our previous cytokine study in SCI where we observed elevated levels of M2 markers such as arginase-1 and IL-10 under Nrg-1 therapy [
13]. M2 macrophages are shown to promote oligodendrocyte differentiation, survival, and remyelination [
62] and are associated with overall tissue preservation and improved recovery of function following SCI [
63]. In the chronically injured spinal cord tissue, however, it was intriguing that Nrg-1 treatment more prominently promoted the population of M1 macrophages with a concurrent increase in IL-10 expressing M2 subpopulation. Further studies are required to fully elucidate the underlying mechanisms of Nrg-1 in macrophage regulation.
We show that Nrg-1 treatment provided a more balanced chemokine profile after SCI. Nrg-1 elevated the expression level of CCL11 in chronic SCI. CCL11 is produced by microphages/microglia, astrocytes, and pericytes [
64‐
66]. Similar to IL-10, CCL11 is known to have anti-inflammatory function and protects neural tissue during inflammatory process [
39]. CCL11 also increases migration and proliferation of neural precursor cells following cerebral ischemic injury [
43], which is a prerequisite for endogenous cell replacement. We additionally showed that Nrg-1 attenuated the expression of CXCL1 in acute SCI. This chemokine is produced by mast cells, macrophages, and astrocytes and contributes to neutrophil recruitment and exacerbate neuroinflammation following CNS injury [
45]. Interestingly, CXCL1 is known to be regulated by IL-6 [
67], an inflammatory cytokine produced by macrophages, microglia, and astrocytes in SCI [
68,
69]. Thus, these data suggest a correlation between downregulation of CXCL1 and reduction in IL-6 expression by Nrg-1 in our acute SCI studies. Notably, inhibition of IL-6 signaling is associated with reduced glial scarring and improves functional recovery [
70] and neuropathic pain following SCI [
25]. Our chemokine profiling also showed the potential of Nrg-1 in attenuating the expression of CXCL2 and CXCL10. CXCL2 is known to induce neuronal injury in motoneuron cultures [
71] supportive of a neuroprotective role for Nrg-1 in CNS injury. CXCL10, also known as interferon-gamma-induced protein 10, is a pro-inflammatory chemokine produced by macrophages, fibroblasts, astrocytes, and endothelial cells upon IFN-ɣ stimulation [
72]. Neutralization of CXCL10 has been associated with decreased secondary tissue degeneration and improvement of functional recovery in murine SCI [
35]. Taken together, our chemokine analysis indicates that while Nrg-1 promotes the overall recruitment of macrophages into the injured spinal cord, it activates a phenotype in macrophages that can facilitate repair processes following SCI.
A novel finding in our study is the modulatory effect of Nrg-1 on T cells after SCI. We have provided the first evidence that Nrg-1 treatment remarkably enhances a T
reg response in acute, subacute, and chronic SCI. Most interestingly, Nrg-1 exerts its beneficial effects systemically by increasing the population of circulating T
reg cells in the bloodstream in chronic SCI. T
reg cells play pivotal roles in regulating an adaptive immune response and preventing autoimmune reactions [
73]. Ablation of T
reg cells elicits an intensive T
eff response that induces neuronal death following CNS injury [
54]. In our study, we showed the ability of systemic Nrg-1 treatment to suppress the population of pro-inflammatory CD3
+CD4
+IFN-γ
+ effector T cells and reduce IFN-γ
+ within the injured spinal cord tissue at subacute SCI. Of note, IFN-γ is primarily produced by lymphocytes including effector Th1 cells and promotes classic macrophage activation [
29] and neuropathic pain [
24]. Importantly, IFN-γ is implicated in inducing white matter degeneration and necrosis following cerebral ischemia/reperfusion injury and increasing the susceptibility of neurons to apoptosis [
74,
75]. SCI studies have shown that IFN-γ directs its degenerative effects by promoting proliferation of cytotoxic T cells [
8,
76]. Indeed, reduction in IFN-γ expression in subacute SCI under Nrg-1 treatment in this study provides an underlying mechanism by which Nrg-1 ameliorated white matter degeneration in the injured spinal cord in our previous studies [
11,
13]. Nrg-1 also induced a reduction in the expression of CCL5 (RANTES), a pro-inflammatory chemokine produced by astrocytes as well as T cells upon macrophage stimulation [
68,
77]. This observation correlates well with the increased M2 macrophages and decreased population of effector T cells that we observed in our immunophenotypic studies. Increase in CCL5 is implicated in microvascular dysfunction following CNS injury [
42]. Altogether, our observations indicate that Nrg-1 is a positive modulator of T cell response after SCI. Therefore, increasing the deficient levels of Nrg-1 after injury has therapeutic value to augment a T regulatory immune response during the repair processes following SCI.
SCI also elicits a B cell response in the spleen and bone marrow characterized by increased B cell number and elevated serum immunoglobulin levels [
4]. Our findings provide new evidence that Nrg-1 promotes recruitment and regulatory phenotype of B cells in the injured spinal cord with a more pronounced effect at the chronic stage of injury. Similar to T cells, the increase in B cell response mainly represented a rise in the B
reg population in response to Nrg-1 treatment. Interestingly, in contrast to the spinal cord, the number of B cells was initially dropped in the blood after SCI which was independent of Nrg-1 treatment. This observation is consistent with previous reports that showed SCI induces an initial cessation of B cell production in the bone marrow [
23]. There is also evidence that B cells undergo extensive apoptosis following SCI. B cells are the integral component of the adaptive humoral immune response and capable of producing a wide variety of cytokines [
10]. Following SCI, B cells produce autoantibodies against spinal cord tissue that can aggravate the secondary injury processes through complement and Fc receptor (FcR)-dependent mechanisms [
21]. Similar to other immune cells, phenotype and function of B cells can be regulated by the signaling molecules and cytokines available in their microenvironment [
10]. B
reg cells support repair processes as they can suppress activation of helper T cell and their production of TNF-α and IFN-ɣ as well as TNF-α production by monocytes [
78‐
80]. Importantly, B
reg cells play essential roles in the formation and maintenance of T
reg cell population [
81]. Moreover, B
reg cell-mediated IL-10 production has been shown to limit tissue damage and improve recovery of function in a murine model of brain ischemia [
22]. For the first time, we uncovered that Nrg-1 attenuates antibody deposition in the injured spinal cord. Our analysis of IgG and IgM showed that Nrg-1 was mainly effective subacutely. It is plausible that Nrg-1-mediated reduction in the spinal cord levels of IgM and IgG in subacute SCI reflects an indirect role for Nrg-1in modulation of vascular permeability. We have previously shown that Nrg-1 treatment attenuates the activity of matrix metalloproteinase (MMP)-9 in the injured spinal cord tissue [
13]. MMP-9 is involved in the disruption of blood-spinal barrier [
3]. Moreover, IL-6 can increase antibody production by B cells [
82]. Thus, the Nrg-1-induced decline in IL-6 expression in our acute SCI studies can be another underlying cause for the reduced antibody deposition in the injured spinal cord at the subacute stage without alteration in B cell number. Our immunohistochemical analysis showed an increasing trend in IgG deposition in the spinal cord tissue with Nrg-1 treatment at chronic (42-day) time-point. This observation may reflect the modulatory effect of Nrg-1 on B cells that reside chronically in the spinal cord tissue. Our analysis of spinal cord B cells showed a close correlation between increased B
reg population in Nrg-1-treated animals and a trend towards higher production of IgG within the chronically injured spinal cord. This is in line with other studies that have attributed IgG production to B
reg cells [
83]. Interestingly, IgG deposition by B
reg cells has been associated with beneficial immunomodulatory roles that include neutralizing harmful antigens from the microenvironment, inhibiting macrophages and dendritic cell activation, and enhancing the clearance of apoptotic bodies that contain self-antigens [
83]. Moreover, studies by Nguyen and colleagues have also shown a positive role for IgG in recovery from SCI [
84]. These studies revealed that systemic IgG administration increased IgG deposition in the injured spinal cord, which was associated with improved neural tissue preservation and functional recovery in a rat model of cervical SCI [
84]. This evidence suggests that increased level of IgG may exert beneficial effects in SCI. Of note, our previous studies identified that Nrg-1 treatment improves tissue preservation in chronic SCI [
13] that could be attributed, at least in part, to the increase in B
reg cells and IgG production.