Appearance of CLN-5+ leukocytes in blood and CNS during EAE
To get a sense of how disease progression was accompanied by appearance of CLN-5-bearing leukocytes, the total number of CD45+CLN-5+ cells in both blood and CNS compartments was determined, as was their percent representation in each tissue (Fig. 2). Representative flow plots from which these data were derived are further presented in Additional files 2, 3, 4, 5, 6, 7, 8: Figs. S1–S7. Brain and spinal cord were processed together to yield total CNS leukocytes, and flow cytometry performed to identify the CLN-5+ cells.
The amount of CD45+CLN-5+ cells in blood peaked early in the disease course, showing a dramatic increase at D6, the earliest time-point measured, then falling sharply at D9 to a value near that of naïve mice for the remainder of disease. The situation was reversed in the CNS, with the peak number CD45+CLN-5+ cells in the time course detected at D15 EAE. This delay in appearance of CD45+CLN-5+ cells in the CNS is consistent with the transit of CLN-5+ leukocytes from the circulation into the brain and spinal cord during the neuroinflammation that accompanies EAE. That control animals receiving adjuvant alone did not show similar elevation of CD45+CLN-5+ cells—behaving more like naïve animals—argues the appearance of these cells is specifically linked to MOG-induced disease.
The percent of CD45+ cells in blood that were CLN-5+ was highest in naïve mice, dropping steeply as early as D6. After D6, the percent CLN-5+ cells decreased yet more by D9, remaining at this low level until D15. Notably, CLN-5+ cells never reached, on average, more than 0.4% of the CD45+ cells in blood during disease. The situation in CNS was vastly different. The percent of CD45+ cells in CNS that were CLN-5+ showed a dramatic peak early in disease at D6. This level then precipitously declined by D9, and remained near constant until D15. Of further distinction is that 20% of CD45+ cells in CNS were CLN-5+ at D6, 50 × the value seen in blood at this time-point. Such a vastly disproportionate accumulation of CLN-5+ cells in the CNS is consistent with a preference for CLN-5+ rather than CLN-5− leukocytes to extravasate from the blood and enter the brain and/or spinal cord early in EAE pathogenesis.
Contribution of leukocyte subtypes to the CLN-5+ population
After confirming the EAE-associated wave of CLN-5+ leukocytes in the blood, and its subsequent advance into the CNS, the next question was: is bearing CLN-5 a general leukocyte property during EAE, or do particular subtypes show a preference for this action? Utilizing qualitative immunofluorescence microscopy, Mandel et al.  described CLN-5 staining mainly on B cells, T cells and monocytes from normal human blood, and further detailed by qRT-PCR and Western blotting upregulation of CLN-5 RNA and protein, respectively, in total blood leukocytes from MS patients experiencing relapse. To see if there were any parallels with human disease, we analyzed these leukocyte subtypes in blood and CNS by flow cytometry at the various EAE time-points. Additionally, neutrophils were also evaluated for comparison. All the different leukocytes examined showed some segment of their respective populations to be CL5+ by flow cytometry, though the timing of their appearance and relative contributions varied by both subtype and tissue compartment.
CLN-5+ B cells were detected in both blood and CNS compartments (Fig. 3). The number of CLN-5+ B cells peaked in the blood early, at D6. Given the direction of leukocyte movement from blood-to-CNS, the peak number of CLN-5+ B cells in the CNS expectedly occurred later, with the greatest number seen at D15. In blood, the percent CLN-5+ cells remained nearly constant, fluctuating around 1%. In CNS, the percent CLN-5+ cells peaked early on D6 at ~ 25%, then decreased. As was the case for total CD45+ cells, the proportion of B cells that were CLN-5+ was much greater in the CNS than in the blood, possibly reflecting a predisposition for CLN-5+ versus CLN-5− B cells to extravasate into parenchymal brain and/or spinal cord tissue. It might further reveal that CLN-5+ B cells are the pioneering B cells that first enter the CNS, structurally clearing the way for CLN-5− B cells to follow. The continued extravasation of CLN-5+ cells among a far greater number of CLN-5− B cells would still result in expansion of the CLN-5+ B cell population in the CNS but at a reduced proportion. Alternatively, some deposition of CLN-5 protein onto the surface of initially CLN-5− cells might occur during the midst of paracellular diapedesis, as this process has been reported to be accompanied by disruption and remodeling of TJ proteins [31, 32].
CLN-5+ T cells (CD3+) were also observed in both blood and CNS (Fig. 4). However, the pattern of appearance of CLN-5+ T cells in blood was more delayed than that for B cells, peaking later at D12. Their appearance in CNS mirrored that for B cells, being maximal at D15—after peak appearance in blood—again consistent with movement of CLN-5+ cells from blood-to-brain/spinal cord. The percent of T cells in blood that were CLN-5+ showed no overt difference throughout the time course of disease—varying only slightly from 0.04% to 0.08%—but, unexpectedly, was highest in naïve mice, and then dropped precipitously during EAE. This level, moreover, never surpassed more than 0.08% at any time-point during disease—far below the nearly 1.0% level observed for B cells. In contrast to the situation in blood, the percent of CLN-5+ T cells in the CNS did show a peak at D6, as was also the case for B cells. Likewise, the percent CLN-5+ T cells in CNS at this time-point, ~ 14%, was 280× that found in blood—again perhaps reflecting a propensity for CLN-5+ leukocytes—across varied subtypes—to enter the CNS first, leading the way for the preponderance of CLN-5− cells to follow.
The CLN-5+ T cell population was further resolved into CD4 and CD8 subtypes, as both contribute variably to CNS inflammation during EAE [33,34,35,36,37]. The appearance of CLN-5+ CD4 cells in both blood and CNS virtually mirrored that observed for total T cells, peaking in the former at D12, and in the latter at D15. CLN-5+ CD8 cells were only somewhat different, with the highest level of cells detected in blood at D9. CLN-5+ CD8 cells, like their CD4 counterparts, were observed to be at the highest level in the CNS at D15, once again, trailing the peak seen in the blood. The percent of both CLN-5+ CD4 and CD8 cells in blood showed no drastic fluctuations throughout the time course—though CD8 cells showed the biggest difference between D6 and D9—and each of the two T cell subtypes showed a drop during disease from that seen in naïve mice. The percent of both CLN-5+ CD4 and CD8 cells in CNS was highest at D6, though the value for CD4 cells was much lower and showed high variability. It is thus likely that CD8 cells, rather than CD4 cells, contributed more to the spike in percent total T cells in CNS being CLN-5+.
Like B cells, monocytes showed a peak number in blood at the earliest time-point, D6, while falling to a low at the last time-point, D15 (Fig. 5). And they too showed the inverse in the CNS, being maximal at D15, and minimum at D6. The percent of blood monocytes that were CLN-5+ was highest in naïve mice (0.35%), as was the case with T and B cells, but then gradually dropped at D6, and then fell even more at D9, from there on remaining near constant to D15 (0.1%). The proportional representation of CLN-5+ monocytes in blood was thus lower than that for B cells but higher than that for T cells. Fluctuations in the percent CLN-5+ monocytes in the CNS showed a similar scenario to that seen in blood, being highest at D6 (10%), then dropping at D9 and continuing near invariant through D15 (~ 2.5%). But, as with B cells, T cells and CD45+ cells in general, the maximum percent CLN-5+ monocytes in CNS was higher than that in blood by more than one order of magnitude, revealing a disproportionate association of CLN-5 with monocyte penetration of the BBB.
CLN-5+ monocytes were further sorted into two functional subsets characterized by distinct migratory and inflammatory properties: Ly6Clo non-inflammatory monocytes, which display CXCR3-mediated recruitment and serve in a vascular patrolling capacity ; and Ly6Chi inflammatory monocytes, which rapidly migrate into inflamed tissues in a CCR2-dependent manner , produce inflammatory cytokines , and are associated with an earlier onset and increased severity of EAE . Separation in this way was meant to see if acquisition of CLN-5+ could be related to inflammatory status. In blood, the two monocyte subsets showed notably different appearances of CLN-5+ cells. The number of non-inflammatory monocytes peaked early at D6, then dropped sharply at D9 to near naïve level, and returned to naïve level by D15. In contrast, inflammatory monocytes gradually accumulated in blood during the course of EAE, steadily rising from the level found in naïve mice to a peak at D12, and then returning to normal by D15. Yet, despite their very dissimilar scenarios in blood, the two monocyte subsets displayed comparable appearances of CLN-5+ cells in CNS, both showing a small spike at D9, a brief return to normal, and then a peak at D15. The percentages of CLN-5+ cells among the respective non-inflammatory and inflammatory monocyte populations in blood were also comparable in both chronology and degree, being maximal in naïve animals (0.4% and 0.2%, respectively), dropping to < half these values at D6 EAE, and then hovering around a near constant value (< 0.1%) through D15. Likewise, non-inflammatory and inflammatory CLN-5+ monocytes displayed similar frequencies in CNS during EAE. Specifically, the percentages of CLN-5+ cells among both monocyte populations were slightly higher than naïve levels at D6 (ranging from ~ 8.6% [non-inflammatory]—15% [inflammatory]), dropping more than half in each case by D9, and then remaining at ~ these levels until D15. As with total monocytes, the peak percentages of both non-inflammatory and inflammatory monocytes in CNS were much greater than those found in blood—though there was considerable disparity between the two populations. The peak percentage of non-inflammatory monocytes in CNS was ~ 37.5× that for blood, while that for inflammatory monocytes was ~ 300× the corresponding blood value. A possible inference for this distinction may be that inflammatory monocytes are more dependent on CLN-5 for BBB transit than are non-inflammatory monocytes.
Though Mandel et al.  did not report neutrophils as being a prominent leukocyte subtype bearing CLN-5 protein in blood of healthy humans or those with MS, these cells were examined here as they enter the CNS and contribute to pathogenicity during EAE [42, 43] (Fig. 6). As with B cells and monocytes, the respective time courses of number of neutrophils in blood versus CNS were mirror images of each other, being highest in the blood at D6 and lowest at D15, while being lowest in the CNS at D6 and reaching a peak at D15. Similar to that found with B and T cells, the percent of CLN-5+ neutrophils in CNS was clearly highest at D6 (40%) and much greater (100×) than the percent detected in blood (at most, 0.4%), which likewise peaked at this early time. Of all the leukocyte subtypes, neutrophils achieved the highest percent CLN-5+.
Leukocyte CLN-5+ mean fluorescence intensity (MFI) varies during EAE
In addition to changes in the total and relative number of CLN-5+ leukocytes in blood and CNS, the average, relative amount of CLN-5 protein per cell—reflected by normalized, CLN-5+ MFI values detected by flow cytometry—appeared to fluctuate as well. As all subtypes evaluated contributed to the overall CLN-5+ leukocyte population in blood and CNS, MFIs were determined for each one (Fig. 7).
After an initial drop from the level in naïve animals, CLN-5+ MFI in both blood and CNS trended upward gradually from D6 to D12, then peaked sharply at D15. As at or around this time of disease there is heightened inflammation, when TJs are being broken down and considerable CLN-5 protein is lost from the CNS vasculature [21, 44, 45], B cells may have acquired some CLN-5 protein that was liberated into the circulation, the inter-endothelial cleft, and/or perivascular space through proteolysis or other means [46, 47].
The two T cell subtypes displayed CLN-5+ MFI patterns that were each distinct from that of B cells, as well as from each other. CLN-5+ CD4 cells had the highest MFI in blood at D6, while the MFI of CLN-5+ CD8 cells remained invariant in blood during the time course of disease. The MFI patterns also differed in the CNS. MFI for CLN-5+ CD4 cells peaked at D15—the same time when most CD4 cells appeared in this compartment. By contrast, CLN-5+ CD8 cells showed the highest MFI value earlier, at D9—prior to their greatest accumulation in the CNS. Peak accumulation of CLN-5+ CD4 and CD8 cells in CNS thus occurred at the same time, or after, they achieved their maximal CLN-5+ MFI. Notably, save for one instance (CD4 T cells in blood at D6), MFI values during disease were > to those of naïve animals in both blood and CNS compartments, indicating T cells acquired additional CLN-5 protein in the course of EAE.
Unlike the different MFI patterns seen between the two T cell subsets, those for the respective monocyte subsets were similar in blood and CNS. For both subsets in both tissues, there was an increase in MFI as acute EAE evolved, with the steepest rise seen in non-inflammatory monocytes. This more robust change in MFI in non-inflammatory monocytes may, again, reflect that CLN-5 acquisition is more essential to disease-related activities in this population.
The pattern of MFI for CLN-5+ neutrophils resembled that for B cells and monocytes, trending highest in both blood and CNS at D15.
CLN-5+ intensity is related to leukocyte activation
Independent lines of evidence suggest there may be some connection between leukocyte acquisition of CLN-5 and level of activation. The migration of T cells across the BBB and entry into the CNS is dependent on these leukocytes being activated and expressing the necessary repertoire of adhesion molecules [48,49,50], a requirement that might also exist for other leukocyte subtypes . And, Mandel et al.  reported that activation of human T cells in vitro resulted in upregulation of claudin-1, a member of the same TJ protein family as CLN-5. In light of these relationships, and our finding that, during EAE, CLN-5+ leukocytes appear in the CNS in greater proportion to their presence in blood for all subtypes examined, we further sought to determine if CLN-5 abundance in T cells, monocytes and neutrophils correlated with these leukocytes’ state of activation. To gauge this relationship, CLN-5 fluorescence intensity of individual cells, as determined by flow cytometry, was used as a surrogate measurement for CLN-5 abundance. Likewise, single cell-associated, fluorescence intensity of well-recognized activation markers was used for the respective leukocyte subtypes. The activation marker used for T cells was CD44, as it is upregulated in antigen-primed T cells and considered a key activation marker in EAE [51, 52]. For monocytes, though no activation markers, per se, were employed, the relationship between CLN-5+ intensity and inflammatory status, the latter judged by Ly6C intensity, was investigated. Lastly, CD18 was used to identify activated neutrophils, as it is required for extravasation , and its blockade shown to resolve EAE . A Pearson Product–Moment Correlation Coefficient (r) was determined between intensity values for each of these markers and those of CLN-5+. Correlation analysis was confined to blood, as T cells that have entered the CNS are nearly all activated (Additional file 9: Fig. S8), though only a fraction are CLN-5+. The population of all leukocytes examined in blood, by contrast, displayed a broader spectrum of both activation states and CLN-5 intensities. For T cells, r showed remarkable consistency, ranging from 0.74 to 0.81 across all the time-points, being maximal at D9 and reflecting a strong linear association between CLN-5+ intensity and T cell activation status (Fig. 8).
To further reinforce this association, another marker for T cell activation, CD11c, was evaluated, as it is associated with high migratory potential  and correlated highly with CD44 expression (Additional file 10: Fig. S9). Linear mixed effects models revealed the relationship among intensities for CLN-5, CD44, and CD11c. Specifically, these reflected the percent variance in CLN-5 explained by CD44 and CD11c. The mean change in CLN-5 per unit change in CD44 was 0.82 (95% CI 0.76–0.88, p < 2e−16), reduced to 0.41 (95% CI 0.32–0.50, p = 3.06e−16), when adjusted for CD11c. Similarly, the mean change in CLN-5 per unit change in CD11c was 0.84 (95% CI 0.78–0.91, p < 2e−16), reduced to 0.51 (95% CI 0.41–0.60, p < 2e−16), when adjusted for CD44. The association of CD44 or CD11c with CLN-5 was significantly reduced when the other was included in the model as the two are highly correlated (Pearson correlation coefficient r = 0.81). Consistently, the percent of variance explained by CD44 and CD11c (pseudo-R-squared 0.75) did not significantly reduce by dropping one or the other (pseudo-R-squared 0.66 for CD44 only and 0.70 for CD11c only). A 3D interpolation and surface plot further revealed the linear relationship among CLN-5, CD44, and CD11c intensities, underscoring the connection between CLN-5 acquisition and T cell activation (Additional file 11: Fig. S10).
By contrast to the evident relationship between CLN-5 intensity and activation in T cells, the linear correlation found between monocyte CLN-5 intensity and inflammatory status was not as strong (Additional file 12: Fig. S11). Specifically, r values ranged from 0.038 to 0. 549 across the EAE time-points. This aligns with the concept that “activation” and “inflammation” are separate, measurable parameters of monocytes that could, but do not necessarily, overlap . It also coincides with the observation that different monocyte subsets of varied inflammatory states found in the blood all infiltrate the CNS during EAE .
The situation for neutrophils reflected an even weaker relationship (Additional file 13: Fig. S12). There was no evidence of linear correlation between CLN-5+ intensity and neutrophil activation, the latter reflected by CD18 intensity, with r ranging only from 0.062 to 0.251 across the time course of disease. Notably, CD18 intensity for individual neutrophils varied over 25-fold, yet that for CLN-5 only fluctuated ~ twofold. This could indicate that the ability of neutrophils to augment their CLN-5 status—at least during EAE—is restricted when compared to that of T cells. In support of this possibility, the range of CLN-5 intensity in T cells is much greater than that in neutrophils for all but one time-point during EAE.