All mice used in these studies were purchased from The Jackson Laboratory (Bar Harbor, ME). C3H/HeJ strain was used to generate the Bleomycin-induced systemic sclerosis (SSc) murine model, 129x1/svJ mice were used for anti-GBM nephritis, and NZB/WF1 is a typical spontaneous lupus nephritis (SLN) model. C57BL/6J mice were used as controls and for the in vitro experiments. All mice were housed in a pathogen-free animal facility at the University of Houston, TX, USA. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NAP 2011) and have been approved by the Institutional Animal Care and Use Committee at the University of Houston.

BTKB66 (Pharmacyclics Inc.) was formulated in 0.16M citric acid to a final concentration of 4 mg/ml (28). The drug was administrated by oral gavage at 25 mg/kg or 50 mg/kg per day for 4-8 weeks, as indicated in each experiment. The control group was given 0.16M citric acid using the same schedule. The dose and route of administration was chosen based on previously established concentrations needed for significant inhibition of Btk activity using this drug (28) or its structural analogue ibrutinib (8). The chosen drug, BTKB66, has been shown to be selective for BTK as it targets BTK with an IC50 of 13.3 nM, targeting TEC, BLK, FGR and PTK6 with IC50 values of 195-493 nM, but with absence of targeting of 100 other kinases at IC50 values >1000, including Itk (28). The safety profiles of these inhibitors have previously been reported (29). The salubrious effect of BTKB66 on liver damage has been examined and reported in detail (28).

A murine model that mirrors the inflammation-driven aspects of human SSc was induced by intradermal injection of Bleomycin 100 μg per day over a total of 4 weeks in C3H/HeJ mice. To gauge the preventative and therapeutic effects of BTK inhibition on SSc-like disease, mice were randomly categorized into two groups: 1) BTKB66-4W: the BTK treatment was initiated at the same time as the BLM injection; 2) BTKB66-8W: BTK inhibitor treatment was started 4 weeks after BLM injection, when SSc disease was already present. In both groups, BTKB66 and the vehicle control were administered by oral gavage once per day at the dosage of 25 mg/kg/d, over 4 weeks. At the end of treatment, skin and lung tissue were collected for pathology evaluation, skin thickness measurement, and Collagen IV content determination. The skin and lung collagen content were determined using commercial kits, as detailed in Supplementary Information . The treatment was repeated twice, with a total of 10-15 mice per group.

26-week old female NZB/W F1 mice were randomized into treatment and control groups (n=10 in each group). BTKB66 was administered by oral gavage once per day at the dosage of 25 mg/kg/d, while the control group was given the vehicle buffer. Blood and urine samples were collected once per month for renal function tests and autoimmunity detection. One kidney from each mouse was used for pathology evaluation, and the other was used for flow cytometry analysis.

Thirty-two 129x1/svJ mice were used to induce anti-GBM nephritis, as described (30). In brief, mice were pre-immunized using Rabbit IgG (250μg/20g mouse body weight, Sigma) mixed with CFA (100 μL/20g mouse body weight, Sigma), then, i.v injected with 200 µl rabbit anti-mouse GBM serum on D5. The mice were categorized into BTKB66 low dose (25 mg/kg/d), BTKB66 high dose (50 mg/kg/d), Dexamethasone (Dex, 0.4 mg/kg/d), or the PBS control group (n=6-8, per group). The treatment was started three days after anti-GBM injection. Blood and 24hr urine samples were collected once per week for BUN and 24-hrs proteinuria measurements. Kidneys were subjected to pathology assessment.

B cells were purified from 2-month-old C57BL/6J mice spleens by negative selection using a commercial bead kit purchased from Stem Cells. Bone marrow- derived macrophages (BMDM) were generated from B6 bone marrow cells incubated with 10 ng/ml MCSF for five days. B cells were stimulated with anti-IgM or anti-IgM with different concentrations of BTKB66, while BMDMs were incubated with 100 ng/ml LPS, or LPS with different concentrations of BTK inhibitor (10, 100, 1000 ng/ml) for 24hrs. Activation of B cells and BMDMs were accessed by flow cytometry using the indicated cell surface markers. Pro-inflammatory cytokine levels in the BMDM culture medium were measured using commercial ELISA kits (R&D, USA)

At the end of each experiment, the targeted tissues were harvested for pathology evaluation. For SSc mice, the skin and lung were collected for hematoxylin and eosin (H&E) and Masson Trichome staining, while kidneys from the SLN and anti-GBM nephritic mice were subjected to H&E and periodic-acid Schiff (PAS) staining. For skin tissue, dermal thickness, defined as the thickness of the skin from the top of the granular layer to the junction between the dermis and subcutaneous fat, was measured. A blinded pathologist (JH) read and assessed all histology changes blindly using standard scoring systems. For dermatologic changes, epidermal and dermal inflammation, skin fibrosis, and vasculitis were evaluated. For lung histopathology grading, the severity of inflammatory infiltration, airspace dilation, interstitial fibrosis, and vasculitis were determined. Renal pathology changes were scored using a well-established grading method, which evaluated the histological changes in glomerular and tubulointerstitial lesions, and the severity of crescent formation, as described (30, 31). The scoring criteria are detailed in Supplementary Information .

To analyze renal inflammation, renal infiltrating inflammatory cells were extracted and analyzed using flow cytometry. In brief, one kidney from each SLN mouse underwent flow cytometry analysis. Kidneys were minced with a razor blade and digested with 1mg/ml collagenase type 4 (Cat.# LS004188, Worthington Biochemical Corp, NJ, USA) for 30 minutes at 37°C in a shaking incubator. After incubation, cells were immediately moved onto ice, and the digestion was stopped by adding RPMI media 1640 (Cat. # 12633, Thermo Fisher Scientific Inc, MA, USA). Red blood cells were lysed using RBC lysing buffer (Sigma). Cell clumps were disrupted using a syringe with 22G needle and then forced through a 70 µm filter. For flow cytometric analysis, cells were stained with antibodies against B220, CD3, CD4, CD8, CD11b, CD11c, CD21, CD23, CD25, CD45, CD69, CD80, CD86, F4/80, Gr1 (BD Biosciences, CA, USA). Approximately 50,000 events were acquired from each sample. All samples were run on a NovoCyte flow cytometer (ACEA Biosciences, CA, USA). Analysis was performed using Novoexpress (ACEA Biosciences, CA, USA). Experimental details are included in Supplementary Information .

Kidneys from anti-GBM nephritis mice, and skin and lung tissue from mice with SSc-like disease were subjected to immunofluorescence staining for macrophages. Anti-Iba1 (Wako, Cat.# 019-19741, 1:300) was used to label macrophages, while anti-PCNA (Abcam, Cat.#ab29, 1:200) was used to stain proliferating cells. Briefly, after deparaffinization and rehydration, sections were boiled for antigen-retrieval using Citrate buffer (PH6.0). Then, sections were blocked with 5% goat serum with 0.25% Triton X-100 for 2hrs and incubated with primary Abs overnight at 4°C. Alexa Fluor 488, Goat anti-Rabbit IgG (H+L) and Alexa Fluor 555, Goat anti-mouse IgG (H+L) were added as 2^nd Abs (1;1000) for 2hr at RT. After final washing and mounting, a Nikon confocal microscope was used for imaging. For each section, 10 random fields were selected to count positive staining. Iba1⁺ PCNA⁺ cells are indicative of proliferating macrophages.

All data are expressed as mean ± SD. Comparison between two groups was performed using a parametric Student t-test or non-parametric Mann Whitney U test (when data was not normally distributed), as indicated in the figure legends. Comparison between multiple groups was conducted using one-way ANOVA followed with Tukey’s post-hoc test. P values < 0.05 were considered statistically significant. All statistical analysis was performed using GraphPad Prism (V.10.0, GraphPad, CA, USA).

Compared to normal skin, BLM-SSc skin exhibited a significant increase in dermal thickness, prominent inflammatory cell infiltration, robust deposition of collagen in the skin, and less subcutaneous fat tissue, as shown in Figure 1A . However, both the BTK-4W and BTK-8w treated groups exhibited less skin fibrosis, less collagen deposition, and decreased inflammatory cell infiltration. Pathology assessment indicated that both BTK-treated groups had significant decrease in skin thickness ( Figure 1B ), less inflammatory cell infiltration ( Figure 1C ), and a lower skin fibrosis score ( Figure 1D ). Lower hydroxyproline content was noted after BTK treatment, compared to that of the PBS control group ( Figure 1E , 2-tailed t -test, p <0.05, n= 10-15 each group).

As lung fibrosis is a major cause of death in SSc patients, we also investigated the effect of the BTK inhibitor on lung histopathologic changes, including inflammation, airspace dilation, fibrosis, and vasculitis. As shown in Figure 2 , BTK-treated mice exhibited significantly lower inflammation with fewer inflammatory cell infiltrates compared to BLM-SSc lungs. Since lung fibrosis was not prominent in BLM-SSc mice, we were not able to assess the impact of BTK inhibition on this phenotype. No significant hydroxyproline content differences were noted in the lungs of these mice (data not shown).

Several studies have examined the effect of BTK inhibition on SLN mice and found that BTK inhibition ameliorates renal, skin, and brain disease in MRL/lpr mice (7–10). Taking advantage of the improved selectivity and specificity of BTKB66 used in this study, we extended our studies to another commonly studied SLN murine model: NZB/WF1. Treatment was started at 26 weeks of age, at which point some mice were proteinuric. After 8 weeks of treatment, BTKB66 was associated with less renal damage and renal dysfunction in NZB/WF1 mice as evidenced by decreased 24-hr-proteinuria ( Supplementary Figure 1A ). Consistent with improved renal function, BTKB66 treated mice exhibited significantly lower GN scores compared to the control group (1.0 ± 0.38 vs 2.7 ± 0.57, P =0.02, Supplementary Figures 1C, E ).

To explore the effect of BTKB66 on systemic autoimmunity, serum auto-Ab levels were measured by ELISA and the phenotype of splenocytes was determined by flow cytometry. At the end of treatment, the average spleen to body weight ratio was 0.0024 ± 0.0003 in the treatment group and 0.0046 ± 0.0013 in the control group (p = 0.03), indicating that BTKB66 treatment was associated with ~41% reduction in spleen:body weight ratios compared to the control group ( Supplementary Figure 2A ). Serum total IgG, IgM, and anti-dsDNA levels were compared between the treatment and control groups. Total IgM and IgG levels were similar between the treatment and control groups ( Supplementary Figures 2C, D ). Similarly, we did not find significant differences in autoantibody levels between the two groups. However, there was a trend towards lower IgG anti-dsDNA autoantibody levels in the treatment group ( Supplementary Figures 2E, F )

Anti-GBM nephritis shares similar cellular and molecular downstream mechanisms with lupus nephritis (22), although it results in nephritis that is more acute in nature, and less dependent on systemic autoimmunity. Hence, we also examined the therapeutic efficacy of BTK inhibition on anti-GBM nephritis. As shown in Supplementary Figure 3 , the anti-GBM challenged mice exhibited worse renal function, with significantly higher 24hr-proteinuria (A), increased BUN levels (B), and higher GN/TIN scores (C-E), compared to the BTKB66-treated mice. The BTKB66 low and high dose treatment groups both demonstrated therapeutic efficacy with significantly lower levels of BUN and 24-hr proteinuria levels, with significantly improved GN and TI disease scores, comparable to the therapeutic efficacy seen with dexamethasone treatment.

To investigate the underlying pathogenic mechanism mediated by BTKB66 treatment, we next examined the cell populations in both the kidneys ( Supplementary Table S1 ) and the spleen ( Supplementary Table S2 ) of NZBW F1 mice after 8-week BTK inhibition, using flow cytometry.

Activated CD3⁺ T cell and CD4⁺ T cell numbers in spleen was lower by 36% and 37% respectively in the BTKB66 treated group as compared to the control group (both, p = 0.03, Supplementary Figures 4A, C ). Total CD4⁺ T cell numbers decreased by 13% and total CD8⁺ T cell numbers increased by 37% in the spleen (p= 0.01 and p = 0.04, respectively, Supplementary Figure S4B ). However, we did not detect any difference in B cell numbers or subsets, including total B cells, follicular B cells, and marginal zone B cells, following BTKB66 treatment.

In addition, BTKB66 had a strong inhibitory effect on the numbers of intra-renal activated T-cells and myeloid cells, as detailed in Supplementary Table S1 . Mice in BTKB66 treated group exhibited reduced total CD4⁺ and activated CD4⁺ T cells and reduced CD11b⁺, and CD86⁺ cells. There was no significant difference in the total number of CD45⁺ Gr-1⁺ cells between these two groups. However, CD45⁺Gr1⁺ cells in the treatment group expressed less CD86 than the controls(4.06 x 10³ vs 1.52 x 10⁵, respectively, P <0.01) indicating decreased activation. Additionally, F4/80⁺ and activated F4/80⁺ macrophages were lower in the treatment group (4.60 x 10³ vs 1.07 x 10⁴ respectively, P <0.01)

Collectively, these results indicate that the reduced renal pathology; proteinuria; and splenomegaly in lupus mice following BTK inhibition is associated with significantly lower peripheral and intra-renal T-cell numbers, and myeloid cell activation, although the impact on B-cells and autoantibodies was minimal.

Both B cells and macrophages play critical roles in the development of various autoimmune diseases including SSc and lupus nephritis, and are both impacted by BTK activation (6, 10, 16). The immunomodulatory effects of BTK inhibition on B cells and macrophages was explored following in vitro culture. B cells were purified from 2-month-old C57BL/6J mice spleen by negative selection. One group of B cells was stimulated with anti-IgM or anti-IgM with different concentrations of BTKB66 (10,100, and1000 ng/ml) for 24hrs. As shown in Figure 3 , BTK inhibition reduced BTK phosphorylation in B cells ( Figure 3A ). The percentages of CD86⁺B220⁺, CD80⁺B220, MHC II⁺B220⁺, and CD69⁺B220⁺ cells were significantly reduced after co-culture with BTKB66, in a dose-dependent manner ( Figures 3B, C ). Another group of B cells was stimulated with LPS, with or without BTKB66. As noted above, a dose-dependent suppressive effect of BTKB66 on LPS-induced B-cell proliferation was observed ( Figure 3D ).

BMDMs were generated from C57BL/6J mouse bone marrow cells with 5-days MCSF (10 ng/ml) incubation and then stimulated with LPS (50ng/ml), or LPS with different concentrations of BTKB66 (n = 6 per group). Culture supernatant was collected at 12hrs and 24hrs for pro-inflammatory cytokine detection, and cells were harvested at 24 hrs for FACS. As Figure 4 indicates, the percentage of CD68⁺ BMDMs was increased after 24 hrs LPS stimulation, but it was significantly reduced in all BTK inhibition groups. Similarly, TNF-α and IL-6 levels in cell culture supernatant were reduced following BTK inhibition in a dose dependent manner. However, we did not detect any difference in IL-10 levels among the groups. Together, this data supports the in vitro inhibitory effect of BTK inhibition on both B-cell and macrophage activation, although the in vivo changes in B-cells were not detected.

As Figure 5 (left) shows, Iba1⁺ positive cells and Iba1⁺ PCNA ⁺ cells were increased in the kidneys of anti-GBM nephritic mice treated with PBS (PBS group), while it was significantly lower in BTKB66 treated mice, comparable to the effects of Dex. Similarly, both skin ( Figure 5 , right) and lung ( Figure 5 , middle) tissue from the SSc group exhibited significantly increased infiltration by macrophages and proliferating cells (including proliferating macrophages), compared to that of the control group. With BTKB66 treatment, these cells were significantly lower, both in skin and lung. Collectively, these results suggest that BTK inhibition may significantly reduce target organ infiltration by macrophages, including proliferating macrophages.