Highly selective inhibition of Bruton’s tyrosine kinase attenuates skin and brain disease in murine lupus

Female MRL/MpJ-Fas ^lpr /J (MRL/lpr) mice (3–4 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA), and housed and aged at the Albert Einstein College of Medicine animal facility (Bronx, NY, USA). Once the mice were 8–9 weeks of age, mice were started on medicated chow that provided a daily dose of ~ 10 mg/kg of BI-BTK-1 [15] (n = 12), or comparable control chow (n = 12). BI-BTK-1 was synthesized and provided by Boehringer Ingelheim. The mice were kept on the chow until the time of sacrifice. Monitoring for skin lesions was started at 12–13 weeks of age, and the mice underwent behavioral testing at 17–18 weeks of age. All available mice, or for technical considerations a randomly selected subset, were evaluated in the studies detailed subsequently. Animal studies were approved by the institutional animal care committee.

At the time of killing, mice were perfused with ice cold PBS. Lesional skin and brains were harvested, fixed, paraffin-embedded, and sectioned and stained at the Histology and Comparative Pathology Core at the Albert Einstein College of Medicine. Sections were also stained with hematoxylin and eosin (H&E).

Skin sections were blindly scored based upon a system we have described previously [9]. Briefly, skin sections were assigned two separate scores. The first was for the epidermis (0–5, in increments of 0.5) based upon the severity of interface dermatitis and the thickening of the epidermis. The second score was assigned to the dermis (0–3, in increments of 0.5), scoring the amount of infiltrating cells. The scores were added together providing a total skin score for each mouse, ranging from 0 to 8. Brain sections were assessed for choroid plexus infiltration at the level of the dorsal fourth ventricle, and blindly scored based upon stromal expansion and cellular infiltration (ranging from 0 to 4).

Macroscopic skin lesions were scored blindly by trained observers every 2–3 weeks starting at 12–13 weeks of age, as previously described [9]. Multiple body regions were assessed, and assigned a numerical value based upon erythema, alopecia, skin thickening, and scaling. The scores were adjusted for the degree of involvement and the percent of body surface covered. A score was then assigned to each mouse, ranging from 0 to 72 [9].

Sections were deparaffinized and rehydrated, and subjected to antigen retrieval in a citrate buffer (pH 6) for 5 minutes at 90 °C. The sections were then cooled to room temperature, washed three times with PBS, and incubated for 1 hour at room temperature in blocking buffer (20% normal horse serum, 0.5% Triton, in PBS). Primary antibodies were then added and were incubated at 4 °C overnight. After 14–16 hours of incubation, the slides were washed three times with PBS and incubated with the appropriate fluorescently labelled secondary antibodies for 1 hour at room temperature. The slides were then washed, stained with 4′,6-diamid ino-2-phenylindole (DAPI), mounted, and imaged. Specifically, we simultaneously stained sections for multiple different markers. The first stain panel combined rabbit anti-IBA-1 (1:250, Wako); rat anti-CD4 (1:100, Ebioscience); and AF647 conjugated donkey anti-mouse IgG (1:500, Jackson ImmunoResearch Labs, West Grove, PA, USA). For this stain, the secondary antibodies used were donkey anti-rabbit AF488 (1:200) and donkey anti-rat AF594 (1:200) (Jackson ImmunoResearch). The second stain panel used both rabbit anti-IBA-1 and rat anti-Mac2 (both at 1:100, Ebioscience), with anti-rabbit AF488 and anti-rat AF594 as secondary antibodies (Jackson ImmunoResearch). A third stain panel contained rat anti-CD4 (1:100, Ebioscience); rabbit anti-B220 (1:100, Affymetrix); and goat anti-C3 (1:100, MP Biomedicals). For this stain, the secondary antibodies used were donkey anti-rat AF 594, donkey anti-rabbit AF488, and donkey anti-goat AF647 (all 1:100, Jackson ImmunoResearch). Finally, the fourth stain panel contained rat anti-fibronectin (1:100, Abcam); goat anti-albumin (1:100, Bethyl Laboratories); and AF647 conjugated donkey anti-mouse IgG (1:500, Jackson Immuno Research). For this stain, the secondary antibodies were donkey anti-rat AF488 and donkey anti-goat AF594 (1:100, Jackson ImmunoResearch). To stain for neutrophils, we stained skin and brain sections with rat anti-Ly6G (1:100, BD-Bioscience), and used donkey anti-rat AF488 (1:100, Jackson ImmunoResearch) as the secondary antibody. For assessment of apoptotic cells in paraffin tissue, the In Situ Cell Death Detection Kit, Fluorescein (Sigma) was used according to the manufacturer’s instructions.

MRL/lpr mice spontaneously display a neuropsychiatric phenotype, including cognitive abnormalities (memory deficits) and depression-like behavior. To assess the effects of BI-BTK-1 on brain disease, neurobehavioral testing was performed as previously described [18]. Briefly, in the object placement test mice were exposed to two identical objects in different locations within an arena for 5 minutes. The mice were then removed, and after a 25-minute retention interval returned to the testing arena, where one of the objects had been moved to a novel location. The relative time the mouse spent exploring the objects was measured and the percent preference calculated. In the object recognition test, the mice were put into an arena and allowed to explore two identical objects for 3 minutes. Mice were then removed, subjected to a 90-minute retention period and returned to the arena, where one of the objects had been replaced with a novel object. The relative time spent with the objects was measured, and the percent preference was calculated. Cognitively normal mice (i.e. with intact memory) will exhibit a preference for objects in a new location (object placement test) or a novel object (object recognition test) [18]. To measure depression-like behavior, the Porsolt swim test was used as previously described [18]. Briefly, mice were placed into a tank of water at room temperature. After an adjustment period of one minute, the amount of time spent immobile was measured as an indication of despair and depression-like behavior.

Snap-frozen skin was homogenized in Trizol using a Retsch MM300 Tissue Lyser to collect RNA. Chloroform was then added and the aqueous phase was collected and processed using the Agencourt RNAdvanced tissue kit that was modified for automation on a Biomek FXp from Beckman. RNA was quantified on a NanoDrop 8000 instrument and RNA quality was assessed based on RNA integrity numbers using the Agilent 2200 Tape Station. Reverse transcription was achieved using the TaqMan Reverse Transcription Reagents Kit (Applied Biosystems). The resultant cDNA was used in a ViiA 7 Real-Time PCR system (Applied Biosystems) using mouse-specific probes from Applied Biosystems.

Protein was isolated from the skin using Tper buffer (Thermo Fisher Scientific, Waltham, MA, USA), and the isolated protein then used in Biolegend’s Legendplex Mouse Inflammation Panel (13-plex) per the manufacturer’s instructions.

Brains were harvested from mice perfused with ice cold PBS, and then the choroid plexus of the third, fourth, and lateral ventricles was dissected out and combined. Choroid plexuses were infused with digestion buffer (2.5 mg/mL Liberase TL (Roche) and 1 mg/mL of DNase I (Roche) in Hank’s balanced salt solution (HBSS) plus magnesium and calcium), cut into small pieces and put into C-tubes (Miltenyi). C-tubes were positioned on a MACS dissociator and run on the m_brain_3 protocol, after which they were placed in an incubator for 30 minutes at 37 °C with shaking at 200 rpm. After incubation, C-tubes were positioned back on the MACS dissociator and run on the m_brain_3 protocol. The released cells were then passed through a 40-μm nylon filter with a cell masher, and filters were washed with 50 mL of wash buffer (1% BSA in HBSS plus magnesium and calcium). Cells were stained with a Fixable Viability Dye efluor 506 (eBioscience), incubated with Fc-Block (BD Bioscience) and stained with the appropriate fluorochrome-conjugated antibodies (Additional file 1: Table S1). Cell counts were determined using 123count eBeads Counting Beads according to the manufacturer’s instructions (eBioscience). Data were acquired on a BD LSR II flow cytometer (BD Biosciences, San Jose, CA, USA). Compensation and analysis of the flow cytometric data were performed using Flowjo software (TreeStar, Ashland, OR, USA). “Fluorescence minus one” controls were used when necessary to set up gates. The gating strategy is illustrated in Additional file 2: Figure S1.

Serum IgG levels were measured by ELISA, as previously described [8].

MRL/lpr mice were treated with control chow or chow formulated with the BTK inhibitor, BI-BTK-1, starting at 8–9 weeks of age until the time of sacrifice (~25 weeks of age). BI-BTK-1 treatment significantly ameliorated the skin lesions seen in control mice by 19 weeks of age (Fig. 1a). Furthermore, this protection was maintained until the time of sacrifice, at which point only 5/12 (42%) of the BI-BTK-1 treated mice had any signs of skin disease, whereas 11/12 (92%) of the control mice had visible cutaneous involvement (p < 0.0001) (Fig. 1b). While some BI-BTK-1 treated mice still displayed alopecia or minor erythema, the skin appeared significantly healthier than in the control-treated counterparts (Fig. 1a, c). In contrast, control-treated mice developed severe macroscopic lesions characterized by alopecia, erythema, scaling, and thickening of the skin on both the face and dorsal thorax (Fig. 1c).

Control-treated MRL/lpr mice displayed histopathologic features of CLE, including thickening of the epidermis (hyperkeratosis) and cellular infiltration (Fig. 2a). In addition to alleviating macroscopic lesions, we found that treatment with BI-BTK-1 significantly improved cutaneous histopathology compared to control MRL/lpr mice (Fig. 2b). Evaluation of the blindly scored sections confirmed that BI-BTK-1 treated mice had significantly improved skin architecture compared to control mice (Fig. 2c).

To further characterize the effects of BTK inhibition on spontaneous skin lesions in MRL/lpr mice, sections were stained for commonly infiltrating cells in CLE, namely macrophages (IBA-1⁺) and T cells (CD4⁺), to assess the effect of BTK inhibition on immune cell infiltration. Additionally, sections were stained for IgG to assess the effect of BI-BTK-1 on immunoglobulin deposition in the skin. While we found no significant difference in infiltration of B cells between the groups (data not shown), skin from BI-BTK-1 treated mice exhibited distinctly fewer macrophages, T cells, and IgG deposition compared to control mice (Fig. 3a and b). We also stained for neutrophils in the skin, but found no significant difference between the BI-BTK-1-treated and control-treated groups (data not shown). The intensity of each stain was measured using ImageJ, verifying that BI-BTK-1-treated mice had significantly diminished infiltration of both IBA-1+ macrophages and CD4+ T cells, and attenuated IgG deposition (Fig. 3c). Circulating serum IgG levels were also significantly decreased with BI-BTK-1 treatment (Fig. 3c, right panel). To further characterize the skin-infiltrating macrophages, sections were also stained with Mac-2, a macrophage activation marker. As can be seen in Fig. 3d, not only were there far larger numbers of IBA-1+ macrophages in the lesional skin of control mice, a large majority of them were Mac-2+ as well.

We assessed the relative expression of mRNA for various inflammatory mediators in the skin. As seen in Fig. 4a, control-treated MRL/lpr mice had increased TNF, monocyte chemoattract protein 1 (MCP-1), IL-10, IL-27, and granulocyte macrophage-colony stimulating factor (GM-CSF) as assessed by RT-PCR. However, treatment with BI-BTK-1 significantly reduced the expression of these inflammatory cytokines (Fig. 4a).

We also assessed the levels of various inflammatory cytokines in the skin at the protein level. As seen in Fig. 4b, BI-BTK-1 decreased the levels of a variety of cytokines previously associated with CLE pathogenesis [19], including TNF, IL-6, IL-17A, and IL-10. Additionally, the macrophage related cytokines MCP-1 and GM-CSF were also decreased by BI-BTK-1 treatment (Fig. 4b). Interferon-γ (IFNγ) and IL-1β levels, however, were not affected by BTK inhibition (data not shown).

MRL/lpr mice display cognitive dysfunction, including impaired visual and spatial memory, beginning around 16 weeks of age [18]. We performed behavioral testing on the BI-BTK-1 treated mice and control mice at 17 weeks of age, with mice subjected to both the object placement (spatial memory) and object recognition (visual memory) tests. Treated mice had a significant improvement in the object placement test, indicating preserved spatial memory (Fig. 5a); treated mice had a mean preference for exploration of the object at the novel location of 61 ± 13%, as compared to a preference of 50 ± 15% in the control mice (p < 0.05). A trend toward improvement was also observed in the object recognition test; however, this was not statistically significant (Fig. 5b).

MRL/lpr mice exhibit depression-like behavior as early as 5 weeks of age [18]. The effect of BI-BTK-1 treatment on this behavior was assessed at 17 weeks of age. We found that starting treatment with BI-BTK-1 at 8–9 weeks of age did not improve depression-like behavior, as assessed by the Porsolt forced swim test (Fig. 5c). Open field tests revealed that neither mouse group had gait deficits or musculoskeletal weakness, as both groups displayed similar total track lengths (data not shown). There were no differences in center track length, center time, or center visits, to indicate changes related to anxiety or risk-seeking behavior (data not shown).

We assessed H&E-stained brain sections to determine the effect of BI-BTK-1 treatment on infiltration of inflammatory cells into the central nervous system. Specifically, we focused on the choroid plexus, the site of the blood-cerebrospinal-fluid barrier, where we and others have previously described marked lymphocyte infiltration in the MRL/lpr strain starting at 16 weeks of age [20]. While control-treated MRL/lpr mice have prominent cellular infiltration into the choroid plexus in the region of dorsal fourth ventricle (Fig. 5d), BI-BTK-1-treated mice had marked diminution in the number of infiltrating cells (Fig. 5e). When the sections were blindly scored for stromal expansion and the degree of infiltrating immune cells, we found that BI-BTK-1 treatment significantly reduces choroid plexus pathology as compared to control mice (Fig. 5f). We also evaluated the presence of apoptosis within the cortex, choroid plexus, and hippocampus, but found no significant differences in the number of TUNEL-positive cells between BI-BTK-1-treated and control-treated mice (data not shown). Furthermore, there were no notable differences in markers of blood-brain barrier permeability, as assessed by staining for fibronectin, albumin, and IgG (data not shown).

To further investigate if the improvement in the neurobehavioral phenotype in BI-BTK-1-treated mice is associated with decreased brain infiltration by particular cell types, flow cytometry was performed on cells isolated from the choroid plexus of BI-BTK-1-treated and control-treated MRL/lpr mice age 17–18 weeks. We found that BI-BTK-1-treated mice exhibited significantly decreased infiltrating leukocytes (CD45+ cells), including CD4+ T cells, CD8+ T cells, and CD19+ B cells, as well as macrophages and monocytes (Fig. 6a).

To confirm the flow cytometric results in analyzing whether BI-BTK-1 was preventing the specific accumulation of particular types of immune cells in the choroid plexus, brain sections were stained for macrophages (IBA-1), T cells (CD4), and B cells (B220). We found that BTK inhibition prevented the accumulation of all three cell types (Fig. 6b, c). Furthermore, a decrease in locally deposited IgG and C3 was noted as well (Fig. 6b, c). We assessed the state of macrophage activation in the accumulating IBA-1+ cells by staining for Mac-2. As can be seen in Fig. 6d, not only do the control mice have more IBA-1+ macrophages but a larger percentage are Mac-2 positive, indicating a more inflammatory macrophage phenotype accumulating in the choroid plexus of control-treated mice. Finally, no meaningful accumulation of neutrophils was observed in the choroid plexus of either control-treated or BI-BTK-1-treated mice (data not shown).