IgM Immunoglobulin Influences Recovery after Cervical Spinal Cord Injury by Modulating the IgG Autoantibody Response

IgM-KO mice show increased deposition of complement-fixing IgG antibodies

Antibodies accumulate in the injured parenchyma during the subacute phase of cervical SCI in rats (Ulndreaj et al., 2017) and during the chronic phase of thoracic SCI in mice (Ankeny et al., 2009). Such antibodies have been shown to be autoreactive (Ankeny et al., 2006, 2009; Ulndreaj et al., 2017) and pathogenic by activating the classical complement system pathway (Ankeny et al., 2009). As IgM has been shown to regulate IgG autoimmunity under normal conditions (Boes et al., 2000), we hypothesized that IgM-KO mice would have increased IgG-antibody deposition in the lesioned spinal cord during the subacute phase of our cervical SCI model, compared with their WT counterparts.

In line with this hypothesis, we found enhanced IgG deposition in IgM-KO mice, as shown by increased %area of IgG+ immunofluorescence compared with WT mice (Fig. 1A,B; Table 1). Particularly at distances −1200, −600, and +600 μm from the injury epicenter, the deposited IgG was ∼125% higher in KO mice than in the WT group. However, the enhanced antibody accumulation did not appear to be sustained in the chronic phase of injury, as there was no significant difference in % IgG+ immunofluorescent area between injured IgM-KO and WT mice at 10 weeks post-SCI (Fig. 1C; Table 1).

To establish whether the IgG deposited in the spinal cord after injury could activate the complement system, we selected sections with the highest IgG accumulation (e.g., at distances −1200, −600, and +600 μm from the injury epicenter) and co-stained them for IgG and complement C3b protein (Fig. 1D). As expected, there was increased IgG deposition in IgM-KO mice in comparison to controls (Fig. 1E; Table 2). Moreover, the IgG-C3b co-stained area was significantly larger in IgM-KO mice than in WT controls with SCI, indicating enhanced complement-fixing activity of deposited IgG in the spinal cord of KO mice (Fig. 1F; Table 2). However, C3b deposition alone was not significantly different between IgM-KO mice and WT controls with SCI (Fig. 1G; Table 2) suggesting that lack of IgM does not affect complement deposition through all pathways, but rather complement deposition through the classical (antibody-mediated) pathway. Of note, one cannot exclude the possibility that lack of statistical difference (effect size 12.5, 95%CI: 1.48, 23.1, permutation p = 0.116; Table 2) between the groups in Figure 1G could be because of increased variance in the WT samples.

To exclude Fc-mediated binding of the labeling anti-C3b antibody in the spinal cord, in our optimization assays we used a mix of Fc blocking peptides (Innovex, #NB30930) and compared binding of antibodies to untreated sections. However, we did not find a significant difference in binding of anti-C3b antibody between the samples treated with Fc block and the untreated samples, even in sections of high antibody binding such as the injury epicenter (data not shown). Thus, binding of the anti-C3b antibody in our spinal cord sections was not Fc-mediated.

Total levels of circulating IgG were similar between IgM-KO and WT mice at BSL (24 h before SCI) and at 2 and 10 weeks post-SCI (Fig. 1H; Table 1). This suggests that increased presence of parenchymal IgG deposition in IgM-KO mice at two weeks post-SCI is not because of higher circulating IgG levels in these mice. Moreover, IgG levels increased in both groups at 2 and 10 weeks post-SCI compared with their respective BSL levels (Fig. 1H; Table 2). Lastly, while there was no detectable IgM immunoglobulin in KO mice before and after the injury, serum levels of IgM increased at two weeks following SCI (Fig. 1I; Table 2).

IgM-KO mice have more T-lymphocytes and microglia/macrophages with myelin-engulfing activity in the lesioned spinal cord

Inflammation mediated by microglia/macrophages and T-lymphocytes is a well-known regulator of pathophysiology in SCI (for review, see Donnelly and Popovich, 2008). Thus, we asked whether the absence of IgM in mice with SCI was linked to an enhanced presence of T-lymphocytes and microglia/macrophages. Indeed, IgM-KO mice showed larger CD3+ immunofluorescent areas and CD3+ cell counts, as compared with WT controls, at two weeks post-SCI (Fig. 2; Table 1). At 10 weeks post-SCI there was no difference in CD3+ immunofluorescence area between the IgM-KO and WT groups, but IgM-KO mice showed elevated CD3+ cell counts overall, and especially 1800 μm rostrally from the injury epicenter (Fig. 2C; Table 1).

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Figure 2.

IgM-KO mice have increased T-lymphocyte infiltration compared with WT mice at two weeks post-SCI. A, Representative sections stained for T-lymphocytes marker CD3 and DAPI in IgM-KO and WT mice at two weeks post-SCI. Quantification of % CD3+ immunostained area/total spinal cord section area (B) and CD3 cell counts (C) rostro-caudally from the injury epicenter, at 2 and 10 weeks post-injury in IgM-KO and WT mice. N = 4–5 mice/group (B), 5 mice/group (C), mean ± SEM; *p < 0.05, **p < 0.01 (see Table 1 for detailed statistical results).

In line with our hypothesis, we also found significantly larger CD11b+ %immunostained areas in IgM-KO mice than in WT mice at 2 weeks but not at 10 weeks (Fig. 3A,B; Table 1) of SCI. CD11b+ cell counts were higher in IgM-KO mice at both 2 and 10 weeks after injury (Fig. 3C; Table 1).

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Figure 3.

IgM-KO mice have more microglia/macrophages than WT controls. A, Representative spinal cord sections stained for the microglia/macrophage markers CD11b and DAPI at two weeks post-SCI. Quantification of % CD11b+ immunostained areas/total area (B) and CD11b+ cell counts (C) rostro-caudally from the injury epicenter, at 2 and 10 weeks of injury. D, Representative spinal cord section stained for myelin (fluoromyelin) and microglia/macrophages (CD11b) at two weeks post-SCI. Inset indicates area of co-localized CD11b and fluoromyelin signal. Myelin-engulfing macrophages appear yellow because of colocalization of green (CD11b+ signal) and red (fluoromyelin) signals in the single layer confocal image. E, Gardner–Altman estimation plot showing quantification of % area of co-localized CD11b+ and fluoromyelin+ signal/total section area. Each dot indicates mean as averaged from 3 sections/animal, where sections were taken at distances −1200, −600, and +600 μm from the injury epicenter. Scale bar: 100 μm, N = 4–5 animals/group (B, E), 5 animals/group (C). Error bars indicate SEM; *p < 0.05, **p < 0.01. See Table 1 for detailed statistical results in B, C and Table 2 for statistical results in E.

Next, we asked whether these microglia/macrophages had engulfed myelin, and whether there was a difference between IgM-KO mice and WT mice regarding the extent of myelin engulfment by microglia/macrophages. To this end, we semi-quantified the %area that was co-stained for CD11b and fluoromyelin (Fig. 3D) in sections with the highest IgG deposition (i.e., −1200, −600, and +600 μm from the injury epicenter) at two weeks post-SCI. Our data showed a significant increase in CD11b+fluoromyelin+ areas in the spinal cords of IgM-KO mice compared with WT controls at two weeks of SCI (Fig. 3E; Table 2), suggesting enhanced myelin engulfment by microglia/macrophages in the absence of IgM immunoglobulin.

IgM-KO mice have larger lesion size in comparison to WT mice following SCI

We quantified the volume of lesional tissue and white/gray matter sparing at 2 and 10 weeks post-SCI. Lesioned areas were present in both the gray and the white matter. In the gray matter, lesions had inflammation, apparent disruption of the normal cytoarchitecture along with fibrosis. In the white matter, lesions presented with inflammation, loss of LFB staining and vacuoles (Fig. 4A). There was no statistical difference between the groups in the volumes of lesional tissue (Fig. 4B; Table 1) or white matter preservation (Fig. 4C; Table 1) at two weeks of injury. However, the preserved gray matter was significantly lower in IgM KO mice, particularly at 600 μm rostral from the injury epicenter (Fig. 4D; Table 1).

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Figure 4.

IgM KO mice have more lesional tissue at 10 weeks post-SCI. A, Representative HE and LFB images of spinal cord cross-sections taken from an injured IgM-KO and WT mouse at the injury epicenter and 600 μm rostral from the injury epicenter at 10 weeks post-SCI. Insets 1–4 indicate most severe lesional tissue. Black arrows indicate gray matter lesions and red arrows indicate white matter lesions. Asterisks indicate vacuoles. Scale bar: 100 μm. B–G, Quantification of lesional tissue and white/gray matter preservation by the Cavalieri method at 2 and 10 weeks post-SCI rostro-caudally from the injury epicenter. B–D, Lesional tissue and white and gray matter preservation in IgM-KO and WT mice at two weeks of injury. E–G, Lesional tissue and white and gray matter preservation at 10 weeks of injury N = 4–5/group; mean ± SEM; *p < 0.05, **p < 0.01.

Of note, IgM KO mice had significant loss of neural tissue at 10 weeks post-SCI. Compared with their WT counterparts, IgM-KO mice with SCI had more lesional tissue, especially rostral to the injury epicenter (Fig. 4E; Table 1). Concomitantly, IgM-KO mice had less white matter preservation rostrally and caudally from the injury epicenter (Fig. 4F; Table 1), while both groups had similar gray matter preservation (Fig. 4G; Table 1) at 10 weeks after injury.

IgM deficiency results in impaired locomotor recovery during the chronic phase of SCI

At two weeks post-SCI, IgM-KO mice and their WT counterparts showed similar locomotor and grip strength performance in BMS and grip strength tests, respectively (Fig. 5A; Table 2). However, at later time points, IgM-KO mice with SCI had impaired coordination and hindlimb function compared with their WT counterparts, based on their performance on the CatWalk test (Fig. 5B). In particular, injured KO mice had worse interlimb coordination compared with WT mice at 8 and 10 weeks post-SCI, as indicated by lower % step sequence regularity indices [Fig. 5C; Table 1 (for multiple comparisons across all time points and groups); Table 2 (for estimation statistics comparing WT-SCI to KO-SCI at each time point)]. By 10 weeks of injury, the difference between IgM-KO and WT mice with SCI was striking, with the regularity index ranging between 44–88% in WT mice, and 14–65% in KO mice.

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Figure 5.

IgM deficiency results in impaired coordination during the chronic phase of SCI. A, Gardner–Altman estimation plots of BMS and grip strength scores at two weeks after injury. See Table 2 for statistics details. B, Representative images of locomotion in a sham WT animal as well as a WT and KO mouse with SCI at 10 weeks after injury, as recorded with CatWalk. C, IgM-KO mice with SCI show deteriorated coordination compared with injured WT mice, as indicated by smaller step sequence regularity indices at 8 and 10 weeks after injury. See Table 2 for statistics details between WT and KO mice. D, Hindlimb swing speed is slower in IgM-KO mice with SCI compared with their WT counterparts at 10 weeks of injury (Table 2). E, There is no difference between WT and KO mice with SCI in forelimb swing speed (Table 2). F, Hindlimb print area is significantly smaller in KO mice with SCI, compared with their WT counterparts (Table 2). G, Forelimb print areas are similar between IgM-KO and WT mice with SCI (Table 2). H, Average body speed is similar between IgM-KO and WT mice with SCI (Table 2). Group size: N = 6–12 mice/group. Mean ± SEM, *p < 0.05, ***p < 0.001. Only significant differences between WT SCI and KO SCI groups are indicated with * on the graph, based on results shown in Table 2. For details on statistical analyses considering the effect of genotype, time after injury, and injury status on parameters shown in C–H, see Table 1.

Hindlimb swing speed was significantly impaired in KO mice compared with WT at 10 weeks of injury [Fig. 5D; Table 1 (for multiple comparisons across all time points and groups); Table 2 (for estimation statistics comparing WT-SCI to KO-SCI at each time point)]. Forelimb swing speed was significantly decreased in both genotypes after SCI when compared with their sham counterparts (Fig. 5E; Table 1). However, the absence of IgM did not appear to affect forelimb swing speed, as injured WT and IgM-KO mice showed similar recovery (Fig. 5E; Table 2).

IgM-KO mice had significantly smaller hindlimb print areas than their WT counterparts at 10 weeks after injury (Fig. 5F; Table 2). In both genotype groups, forelimb print area was reduced after injury when compared with their sham counterparts (Fig. 5G; Table 1). However, there was no difference in forelimb print areas between IgM-KO and WT mice with SCI (Fig. 5G; Table 2). Despite the above differences in coordination, hindlimb swing speed and hindlimb print area between the IgM-KO and WT mice with SCI, the average body speed was similar between IgM-KO and WT mice at all time points of the study [Fig. 5H; Table 1 (for multiple comparisons across all time points and groups); Table 2 (for estimation statistics comparing WT-SCI to KO-SCI at each time point)].

IgM deficiency does not result in respiratory tract infections or urinary tract infections (UTIs)

Infections have been shown to negatively affect neurologic recovery in SCI (Failli et al., 2012). Respiratory tract infections and UTIs are common complications in patients with SCI (Garcia-Arguello et al., 2017). As the peripheral immune response is particularly suppressed during the subacute phase of C7/T1 SCI in rats (Ulndreaj et al., 2017), we hypothesized that IgM-KO mice would have increased bacteria in their bladders and signs of pneumonia in their lungs at two weeks post-SCI. To test this hypothesis, we quantified the bacterial load in bladders of IgM-KO and WT mice with SCI and assessed for signs of inflammation in the lungs (e.g., abnormal presence of macrophages, increased numbers of macrophages and abnormal tissue structure) at two weeks after injury. There were no signs of lung inflammation in IgM-KO mice compared with WT mice (Fig. 6A). In addition, we found no significant increase in bacterial counts in the bladders of IgM-KO mice compared with WT mice with SCI (Fig. 6B; Table 2). Similar microbial counts were found in gut cultures (isolated from the colon), which were used as a positive control for detection and culturing of bacteria (Fig. 6C; Table 2). Other important metrics of recovery from SCI (Brommer et al., 2016) such as body weight (Fig. 6D; Table 2) and spleen weight (Fig. 6E; Table 2) were also similar between IgM-KO and WT mice at two weeks after injury.

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Figure 6.

There is no evidence of bladder infection or pneumonia in IgM-KO mice or WT controls at two weeks of SCI. A, HE-stained lungs at 4× magnification indicating absence of inflammation. B, C, Gardner–Altman estimation plots of bladder-bacterial and gut-bacterial load in IgM-KO and WT mice at two weeks of SCI. Gardner–Altman estimation plots of body weight (D) and spleen weight (E) in IgM-KO and WT mice at two weeks post-SCI. CFU = colony forming units; NS = nonsignificant. See Table 2 for detailed statistical results.

Discussion

This study shows that deficiency of IgM immunoglobulin results in impaired recovery following cervical SCI in mice. Overall, IgM-KO mice exhibited worse neurobehavioral recovery, coupled with increased lesion size, less white matter sparing, and enhanced deposition of complement-fixing IgG antibodies in the spinal cord as compared with their WT counterparts. These data provide evidence for the necessary role of IgM immunoglobulin in spontaneous recovery during cervical SCI and warrant further research into the therapeutic effect of IgM administration after SCI. A recent clinical trial showing significant benefits of IgM-enriched immunoglobulin preparation for patients with pneumonia and lower levels of endogenous IgM (Brunner et al., 2013; Cavazzuti et al., 2014; Wand et al., 2016; Welte et al., 2018; Kalvelage et al., 2019; Willuweit et al., 2019) further supports this notion.

Using a rat model of cervical SCI, we previously found early extravasation of IgM immunoglobulin in the lesioned spinal cord and a chronic expansion of splenic IgM plasma cells (Ulndreaj et al., 2017). Additionally, we have observed chronically increased levels of serum IgM immunoglobulin in rats after cervical SCI (Ulndreaj et al., 2020). Circulating IgM immunoglobulin comprises the majority of naturally occurring antibodies. By binding a broad range of self and non-self-targets, naturally occurring antibodies constitute a rapid innate response to altered self-antigens and invading pathogens, thereby maintaining homeostasis against autoimmunity, infectious and other diseases (Ehrenstein and Notley, 2010). Thus, we hypothesized that the enhanced IgM response we observed in our rat SCI model could be protective. To test this hypothesis we induced C6/C7 SCI in mice that lack secretory IgM immunoglobulin (Boes et al., 1998b) and compared their outcomes to WT littermate controls. These IgM-KO mice have undetectable serum IgM, although they express membrane IgM and all other secretory immunoglobulin classes (Boes et al., 1998b).

IgM-KO mice had significant deficits in coordination and other gait parameters in their hindlimbs compared with their WT counterparts during the chronic time points of our study (8–10 weeks post-SCI). When compared with shams, mice with SCI showed overall impairment in coordination, swing speed and print area in both fore- and hindlimbs, thus confirming the impairments previously observed in our C6/C7 SCI mouse model (Forgione et al., 2017). When comparing IgM-KO to WT mice with SCI, KO mice had greater impairments in coordination, lowered swing speed and smaller print area in the hindlimbs. Importantly, both groups had similar average body speeds, suggesting that the above differences did not result from the confounding effect of body speed (Batka et al., 2014).

In line with these chronic neurologic impairments, we found increased lesion size and less white matter sparing in IgM-KO mice compared with WT mice at our last chronic time point of the study (10 weeks after injury). Interestingly, both groups had similar neurobehavioral and histologic outcomes at two weeks of injury. This suggested that IgM deficiency may affect pathomechanisms that are initiated during or after the two-week time point, which marks the beginning of the subacute phase in rodent SCI.

One relevant mechanism explored here is the expansion of T cells and microglia/macrophages in the injured spinal cord. CD3 and CD11b cell expansion was assessed by quantifying the percent positive immunofluorescent area and by cell counts in spinal cord sections across a selected rostro-caudal axis from the injury epicenter. Of note, while microglia/macrophage responses are considered to peak by two weeks post-SCI in mice, this time point marks only the onset of T-cell infiltration in the lesioned spinal cord (Sroga et al., 2003). Also, in patients with SCI T-cell infiltrates are limited in the spinal cord and do not appear until weeks to months after trauma (Fleming et al., 2006). Given the decline in T-cell signal at 10 weeks, it is possible that here we have missed the peak of T-cell infiltration in the spinal cord. Despite this limitation regarding the optimal time point for the detection of CD3, our data show that compared with WT mice, IgM-KO mice had increased % CD3+ and % CD11b+ immunofluorescence area at two weeks post-SCI. At 10 weeks post-SCI, both groups had similar CD3 and CD11b + immunofluorescence areas. Interestingly, CD3 and CD11b cell counts were significantly higher in IgM-KO mice at both 2 and 10 weeks post-SCI. Our finding of higher CD3 and CD11b cell counts in IgM-KO mice with similar total immunofluorescence areas as WT mice at 10 weeks suggests that these cells in IgM-KO mice are on average smaller than in WT mice. This in turn can indirectly provide some information on the activation status of such cells or their subtype. For example, while both microglia and blood-derived macrophages are CD11b+, microglia tend to have a larger surface than blood-derived macrophages (Servet-Delprat et al., 2002). Similarly, IgM KO and WT mice may have different composition of CD3 cell subpopulations (CD4, CD8) with varying cell sizes depending on their activation state (Li and Kurlander, 2010). Taken together, these data show that CD3 and CD11b cells are expanded in the spinal cord of IgM-KO mice compared with their WT counterparts and further suggest that the composition of these cells is likely different between IgM-KO and WT mice.

Moreover, we found that IgM-KO mice had increased IgG antibody deposition in the lesioned spinal cord at two weeks of injury. We and others have previously observed extensive widespread IgG in the lesioned parenchyma in SCI models (Ankeny et al., 2009; Ulndreaj et al., 2017; Zhou et al., 2019). Such antibodies are localized with neurons (Ulndreaj et al., 2017), bind to astrocytes or astrocytic markers (Ulndreaj et al., 2017; Arevalo-Martin et al., 2018; Hergenroeder et al., 2018) or to vascular endothelial cells within the injured spinal cord parenchyma (Zhou et al., 2019). The deposited antibodies can be autoreactive [e.g., IgGs bind to spinal cord components through their F(ab’)₂ portion] or not (e.g., IgGs bind to spinal cord cells through their Fc portion), and can persist throughout the disease course (Ankeny et al., 2009) or peak only during the subacute phase of SCI (Ulndreaj et al., 2017; Zhou et al., 2019). Regardless, there is strong evidence that IgG deposition in the spinal cord is detrimental to recovery after SCI (Ankeny et al., 2009; Zhou et al., 2019). Of note, circulating levels of IgG were similar between IgM-KO and WT mice at all time points of the study. This suggests that increased parenchymal IgG deposition at two weeks post-SCI in IgM-KO mice is likely not because of higher circulating IgG immunoglobulin. Rather, factors that affect IgG extravasation in the tissue [such as blood-spinal cord barrier (BSCB) disruption] and or/turnover are likely to contribute to enhanced IgG deposition in IgM-KO mice at two weeks post-SCI. The potential effect of IgM on BSCB integrity was outside the scope of this study. However, the accumulation of IgG, which appeared largely in areas mostly affected by the physical impact of clip compression (i.e., injury epicenter, anterior and lateral white matter), suggests that the degree of BSCB disruption may be different between IgM-KO and WT mice.

Here, we do not present direct evidence for the binding specificity of the IgG that was detected in the spinal cord. However, in our previous study we showed that circulating antibodies from rats with cervical SCI bind more strongly to cultured astrocytes (a cell type we found was IgG positive in injured spinal cords) than antibodies from uninjured rats. We also found that the spleen mounts a stronger antibody response against spinal cord antigens after injury. Furthermore, we performed competition assays to show that the signal we detect on the spinal cord is specific (Ulndreaj et al., 2017). These indirect lines of evidence suggest that some of the serum antibodies in mice with SCI are specific to spinal cord antigens. Others have also shown that SCI results in the induction of self-reacting antibodies (Ankeny et al., 2006, 2009). It should be noted that these induced antibodies comprise the minority of circulating antibodies (in the range of μg/ml) and we do not think that the signal we see in the spinal cord results exclusively from induced antibodies. Rather, given the large signal detected, we think it represents binding of naturally occurring antibodies that comprise the majority of the antibody pool in the body (in the mg/ml range), and typically bind to self-antigens with low affinity (Lutz, 2007). Such antibodies are elevated following SCI (Arevalo-Martin et al., 2018). Moreover, it has been shown that in the absence of circulating IgM immunoglobulins, there are more self-reacting IgG antibodies (Boes et al., 2000). Taken together, we think the deposition of IgGs in the injured spinal cord in the present study results from at least three non-mutually exclusive phenomena: (1) the BSCB disruption which allows for serum IgGs to enter the parenchyma non-specifically; (2) the binding of naturally occurring antibodies to spinal cord self-antigens with low affinity; (3) the generation of immune auto-IgGs following SCI and potential exacerbation of this response because of the absence of secretory IgM (Boes et al., 2000) specifically.

The histology and inflammation data taken together indicate that enhanced neuroinflammation (rather than worse lesion) during the subacute phase (two weeks post-SCI) is linked to chronic neurobehavioral impairments in IgM-KO mice. This observation is in line with multiple studies linking early neuroinflammation to deteriorated histologic and locomotor outcomes in the chronic phase (Bao et al., 2011; Nguyen et al., 2012; Brennan et al., 2015). It is unclear why IgM-KO and WT mice have similar lesion size at two weeks despite significant differences in neuroinflammation at the same time point. However, a plausible explanation could be that the enhanced inflammation we observed in IgM-KO mice has long-term functional effects that have not been established by two weeks after injury. These effects could be related to processes including regeneration and plasticity (Ballermann and Fouad, 2006; Courtine et al., 2008; Rossignol and Frigon, 2011), and a favorable balance of remyelination/demyelination (Hesp et al., 2015). Such mechanisms, although initiated early on during SCI, take longer to establish and to give rise to quantifiable differences in neurobehavioral outcomes.

One of the mechanisms by which deposited IgGs have been shown to negatively impact outcomes after SCI is through activation of the complement system (Ankeny et al., 2009). In turn, complement activation enhances immune cell infiltration/activation (such as microglia/macrophages and T-lymphocytes; Ankeny et al., 2009; Brennan et al., 2015) and promotes degradation of spared tissue by phagocytosis (Ankeny et al., 2009) or by direct complement-mediated attack (Qiao et al., 2010). Here, we examined whether increased IgG deposition in IgM-KO mice led to enhanced complement fixation in the lesioned spinal cord. In line with this hypothesis, we found enhanced C3b-IgG co-localization in the spinal cord of IgM-KO mice, as compared with their WT counterparts. Interestingly, C3b opsonizes antigens that are further taken up and engulfed by phagocytic cells (such as microglia and macrophages) through the interaction of C3b and complement receptor 3 (CR3), consisting of CD11b and CD18 (Ricklin et al., 2010). In an effort to understand the source of increased myelin loss in IgM-KO mice with SCI, and given the widespread presence of CD11b+ cells (typically microglia/macrophages) at two weeks of injury in KO mice, we further hypothesized that myelin was engulfed by CD11b+ microglia/macrophages in IgM-KO mice. Indeed, by quantifying the CD11b+/fluoromyelin+ area in sections with maximum IgG deposition (shown to have increased C3b deposition), we found that IgM-KO mice had significantly more myelin-engulfing microglia/macrophages than their WT counterparts. Taken together, this line of evidence indicates that the absence of secretory IgM immunoglobulin is associated with increased complement-fixing IgGs on the lesioned spinal cord, as well as the widespread presence of myelin-engulfing microglia/macrophages.

Research indicates that secretory IgM protects against detrimental IgG autoimmunity in multiple potentially overlapping pathways, which range from regulating the development of IgG autoantibodies to directly inhibiting their effector functions. For example, IgM antibodies enhance the phagocytic clearance of apoptotic cells and other cellular debris (Chen et al., 2009), which if left uncleared, could trigger pathogenic autoimmune responses (Henson, 2017). In addition, IgM regulates the development of pathogenic IgG autoantibody responses by controlling the generation of autoreactive B cells throughout their life span. Such mechanisms include central tolerance to self-antigens early during B cell development (Nguyen et al., 2015), and later on the reduction of autoreactive BCR signaling (Tsiantoulas et al., 2017) and deletion/deactivation of self-reactive B cells (Ouchida et al., 2012). Moreover, IgM immunoglobulin inhibits IgG autoantibody effector functions by binding directly to IgG’s F(ab’)₂ component, thereby inhibiting IgG binding to self-antigens (Adib et al., 1990). We tested the latter possibility, given that in a previous study we found significant deposition of IgM antibodies in the spinal cord following C7 SCI in rats (Ulndreaj et al., 2017). However, contrary to our rat data, we did not find any significant IgM deposition at 2 or 10 weeks of injury in injured WT mice (data not shown), suggesting that IgM does not inhibit IgG binding in the injured spinal cord.

Of note, IgM’s protective effects on SCI recovery could be independent of IgG. For example, IgM was shown to induce proliferation of oligodendrocyte-precursor cells (OPCs) via Fcα/μR signaling during brain development (Tanabe and Yamashita, 2018). Occurring within the first two weeks post-SCI, and primarily rostrally from the injury epicenter (Li and Leung, 2015), oligodendrogenesis is an endogenous regenerative process in rodent SCI (Zai and Wrathall, 2005; Lytle and Wrathall, 2007; Tripathi and McTigue, 2007; Lytle et al., 2009; Li and Leung, 2015) and has been a therapeutic target for multiple research groups aiming to reverse myelin loss after injury (Karimi-Abdolrezaee et al., 2006; Plemel et al., 2014; Papastefanaki and Matsas, 2015). Thus, future studies could investigate whether secretory IgM promotes endogenous OPC proliferation in SCI.

Importantly, circulating IgM is the carrier and regulator of AIM (also known as CD5L/Spalpha/API6; Hiramoto et al., 2018), a protein with debris clearance and macrophage survival function. In the absence of secretory IgM, circulating levels of AIM are undetectable and insufficient debris clearance was shown to lead to worsened disease progression in a model of acute kidney injury (Arai and Miyazaki, 2018). It is thus possible that worse outcomes observed in IgM-KO mice with SCI in the present study are because of insufficient AIM-mediated debris clearance. This possibility should be explored in future studies as AIM was shown to be a top gene upregulated by microglia/macrophages after SCI (Zhu et al., 2017), yet its role in SCI remains unknown.

In addition to the above positive effects, IgM protects against microbial infections. Here, we looked at a possible link between IgM deficiency and microbial infections after injury, as infections are linked with poor neurologic recovery in SCI (Failli et al., 2012; Brommer et al., 2016) and IgM-KO mice have been shown to be susceptible to microbial infections in past studies (Baumgarth et al., 2005). As UTIs and pneumonia are the most frequent infection types in patients with SCI (Garcia-Arguello et al., 2017), we looked at microbial loads in the bladder and signs of inflammation in the lungs at two weeks after injury. However, we found no signs of bladder infection or lung inflammation. Thus, our data suggest that the impaired recovery after SCI in the absence of secretory IgM immunoglobulin is not because of infections. However, it is important to note that our experiments were conducted in mice that were receiving antibiotics in their water and were infection free.

Although our data suggest that total IgM immunoglobulin may have a protective role in SCI, it is important to note that not all IgM is beneficial for SCI. For example, some natural IgM antibodies were shown to have significant detrimental effects on SCI outcomes by activating the immune system and further enhancing secondary injury (Narang et al., 2017). This duality of the IgM response during SCI reflects the overall complex role of the immune response in SCI pathophysiology, which has made the journey to discovering effective SCI therapies challenging. Delineating the role of critical pathophysiological factors in SCI recovery, such as important components of the immune system, will take us closer to developing effective treatments for this debilitating chronic condition, which given its complex nature, will ultimately necessitate the application of an array of treatment modalities (Ulndreaj et al., 2016).

In conclusion, the present study shows that IgM immunoglobulin is important for recovery following cervical SCI, as IgM deficiency resulted in impaired neurobehavioral and histologic outcomes, as well as increased IgG deposition, complement activation, T- cell infiltration and myelin-engulfing macrophages. We anticipate that future research will investigate whether circulating IgM levels are useful biomarkers of recovery in patients with SCI, and ultimately, whether modulating IgM immunoglobulin levels can improve recovery.