Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 DOI 10.1186/s13075-017-1500-0 RESEARCH ARTICLE Highly selective inhiition of Bruton s tyrosine kinse ttenutes skin nd rin disese in murine lupus Smnth A. Chlmers 1, Jing Wen 1, Jessic Doerner 1, Ariel Stock 1, Crl M. Cud 2, Hdijt M. Mkinde 2, Hrris Perlmn 2, Todd Bosnc 3, Deorh We 4, Gerld Nozny 4, Jy S. Fine 4, Elliott Klein 4, Meer Rmnujm 4 nd Chim Puttermn 1,5* Open Access Astrct Bckground: Systemic lupus erythemtosus (SLE) is systemic utoimmune disese tht ffects different end orgns, including skin nd rin. We nd others hve previously shown the importnce of mcrophges in the pthogenesis of cutneous nd neuropsychitric lupus. Additionlly, utontiodies produced y utorective B cells re thought to ply role in oth the skin nd centrl nervous system pthologies ssocited with SLE. Methods: We used novel inhiitor of Bruton s tyrosine kinse (BTK), BI-BTK-1, to trget oth mcrophge nd B cell function in the MRL-lpr/lpr murine model of SLE, nd exmined the effect of tretment on skin nd rin disese. Results: We found tht tretment with BI-BTK-1 significntly ttenuted the lupus ssocited cutneous nd neuropsychitric disese phenotypes in MRL/lpr mice. Specificlly, BI-BTK-1 treted mice hd fewer mcroscopic nd microscopic skin lesions, reduced cutneous cellulr infiltrtion, nd diminished inflmmtory cytokine expression compred to control mice. BTK inhiition lso significntly improved cognitive function, nd decresed ccumultion of T cells, B cells, nd mcrophges within the centrl nervous system, specificlly the choroid plexus. Conclusions: Directed therpies my improve the response rte in lupus-driven trget orgn involvement, nd decrese the dngerous side effects ssocited with glol immunosuppression. Overll, our results suggest tht inhiition of BTK my e promising therpeutic option for cutneous nd neuropsychitric disese ssocited with SLE. Keywords: Systemic lupus erythemtous (SLE), Neuropsychitric lupus (NPSLE), Cutneous lupus erythemtosus (CLE), Bruton s tyrosine kinse (BTK) Bckground Systemic lupus erythemtosus (SLE) is multifctoril utoimmune disese, which results in different end orgn pthologies including prominent skin nd neuropsychitric mnifesttions [1]. Cutneous disese, known s cutneous lupus erythemtosus (CLE), occurs in up to 75% of ptients nd hs profound effect on the ptient s qulity of life [2]. Neuropsychitric SLE (NPSLE) occurs in out 40% of ptients; rin involvement mnifests through diverse rry of signs nd symptoms * Correspondence: chim.puttermn@einstein.yu.edu 1 Deprtment of Microiology nd Immunology, Alert Einstein College of Medicine, Bronx, NY, USA 5 Division of Rheumtology, Alert Einstein College of Medicine, F701N, 1300 Morris Prk Ave, Bronx, NY 10461, USA Full list of uthor informtion is ville t the end of the rticle including seizures, psychosis, cognitive dysfunction, nd depression, nd hs significnt prognostic implictions [3]. Current therpies for oth cutneous nd neuropsychitric mnifesttions re ssocited with multiple side effects rnging from mild to severe (e.g. incresed risk of infection nd cncer ssocited with immunosuppressive gents). These less thn idel side-effect profiles of current therpies limit their use. As such, ptients with SLE would enefit from the development of more trgeted tretment options with improved side-effect profiles, nd perhps incresed efficcy s well [4]. In SLE, cutneous nd rin mnifesttions cn pper lone, without the involvement of other trget orgns (e.g. kidney). Moreover, CLE cn e n isolted disese entity, outside the context of systemic lupus. Finlly, The Author(s). 2018 Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License (http://cretivecommons.org/licenses/y/4.0/), which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver (http://cretivecommons.org/pulicdomin/zero/1.0/) pplies to the dt mde ville in this rticle, unless otherwise stted.
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 Pge 2 of 11 therpeutic pproches to one prticulr orgn mnifesttion in SLE re not necessrily pplicle to disese in other orgns. Bsed on ll of these considertions, there remins pressing need to develop therpies for nonrenl lupus mnifesttions s well. Two mjor cell types contriuting to SLE pthogenesis re B cells nd mcrophges [1]. B cells produce utontiodies tht loclize to lupus trget orgns, including oth the skin nd rin in ptients with CLE [5] nd NPSLE [6], respectively. In CLE, immune complexes re deposited t the dermo-epiderml junction where they cn ctivte locl Fc-receptor-ering cells, inititing n inflmmtory cscde with the potentil to cuse locl dmge [2]. Similrly, in NPSLE utontiodies cn e deposited in the rin fter disruption of the lood rin rrier, promoting inflmmtion nd cellulr dmge. Activted mcrophges re lso found infiltrting into these tissues with ctive inflmmtion, nd their effector functions re thought to e importnt to disese pthogenesis [7 10]. Bruton s tyrosine kinse (BTK) is importnt for oth B cell nd mcrophge function [11]. Specificlly, BTK is vitl for B cell development, survivl, nd function (e.g. B cell receptor (BCR) nd toll-like receptor (TLR) signling), while in mcrophges BTK medites Fc receptor nd TLR signling nd mcrophge polriztion [11 14]. These pivotl roles for BTK in oth B cells nd mcrophges indicte tht this enzyme could potentilly e vlule therpeutic trget in different end-orgn pthologies in SLE. BI-BTK-1 is novel, potent, highly selective, nd irreversile inhiitor of BTK, which we previously hve shown to directly inhiit B cell nd myeloid cell ctivtion y in vitro BCR nd FcγR stimultion, respectively [15]. Furthermore, we hve found tht BI-BTK-1 cn prevent kidney disese in nephrotoxic serum nephritis [15]. However, this prticulr inducile model of immune-complex medited nephritis is short term (1 2 weeks), nd only single orgn (the kidney) is ffected. A more chllenging nd cliniclly relevnt ssessment of the potentil of BTK inhiition to modulte lupus would e in dministrtion of n inhiitor over prolonged period of time, in lupus model with multi-orgn involvement. MRL/lpr mice, which re chrcterized geneticlly y defective Fs-medited poptosis, exhiit spontneous systemic utoimmune disese tht mimics humn SLE, including predominnce in femle nimls, circulting nucler uto-ntiodies, nd pthology in multiple end orgns. In ddition to the development of spontneous skin lesions with clinicl nd histopthologic chrcteristics resemling those seen in humn SLE, disese progression in the MRL/lpr strin is ssocited with cognitive dysfunction nd depression-like ehvior. Therefore, while defects in Fs re not custive in humn lupus, the considertions descried mke the MRL/ lpr strin n excellent nd often-used lupus model, not just for the study of lupus nephritis, ut lso of CLE nd NPSLE [16, 17]. For this study, we ssessed the effect of BI-BTK-1 tretment in cutneous nd neuropsychitric mnifesttions in the MRL/lpr mouse model. We treted mice with BI-BTK-1 nd exmined the development of spontneous skin lesions nd ehviorl normlities, to investigte if BTK represents potentil therpeutic trget for these clssic ut often tretment-resistnt lupus trget orgn mnifesttions. Methods Mice Femle MRL/MpJ-Fs lpr /J (MRL/lpr) mice (3 4 weeks old) were purchsed from the Jckson Lortory (Br Hror, ME, USA), nd housed nd ged t the Alert Einstein College of Medicine niml fcility (Bronx, NY, USA). Once the mice were 8 9 weeks of ge, mice were strted on medicted chow tht provided dily dose of ~ 10 mg/kg of BI-BTK-1 [15] (n = 12), or comprle control chow (n = 12). BI-BTK-1 ws synthesized nd provided y Boehringer Ingelheim. The mice were kept on the chow until the time of scrifice. Monitoring for skin lesions ws strted t 12 13 weeks of ge, nd the mice underwent ehviorl testing t 17 18 weeks of ge. All ville mice, or for technicl considertions rndomly selected suset, were evluted in the studies detiled susequently. Animl studies were pproved y the institutionl niml cre committee. Histopthologic ssessment At the time of killing, mice were perfused with ice cold PBS. Lesionl skin nd rins were hrvested, fixed, prffin-emedded, nd sectioned nd stined t the Histology nd Comprtive Pthology Core t the Alert Einstein College of Medicine. Sections were lso stined with hemtoxylin nd eosin (H&E). Skin sections were lindly scored sed upon system we hve descried previously [9]. Briefly, skin sections were ssigned two seprte scores. The first ws for the epidermis (0 5, in increments of 0.5) sed upon the severity of interfce dermtitis nd the thickening of the epidermis. The second score ws ssigned to the dermis (0 3, in increments of 0.5), scoring the mount of infiltrting cells. The scores were dded together providing totl skin score for ech mouse, rnging from 0 to 8. Brin sections were ssessed for choroid plexus infiltrtion t the level of the dorsl fourth ventricle, nd lindly scored sed upon stroml expnsion nd cellulr infiltrtion (rnging from 0 to 4). Assessment of mcroscopic skin lesions Mcroscopic skin lesions were scored lindly y trined oservers every 2 3 weeks strting t 12 13 weeks of ge, s previously descried [9]. Multiple ody regions
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 Pge 3 of 11 were ssessed, nd ssigned numericl vlue sed upon erythem, lopeci, skin thickening, nd scling. The scores were djusted for the degree of involvement nd the percent of ody surfce covered. A score ws then ssigned to ech mouse, rnging from 0 to 72 [9]. Immunofluorescent stining Sections were deprffinized nd rehydrted, nd sujected to ntigen retrievl in citrte uffer (ph 6) for 5 minutes t 90 C. The sections were then cooled to room temperture, wshed three times with PBS, nd incuted for 1 hour t room temperture in locking uffer (20% norml horse serum, 0.5% Triton, in PBS). Primry ntiodies were then dded nd were incuted t 4 C overnight. After 14 16 hours of incution, the slides were wshed three times with PBS nd incuted with the pproprite fluorescently lelled secondry ntiodies for 1 hour t room temperture. The slides were then wshed, stined with 4,6-dimid ino-2-phenylindole (DAPI), mounted, nd imged. Specificlly, we simultneously stined sections for multiple different mrkers. The first stin pnel comined rit nti-iba-1 (1:250, Wko); rt nti-cd4 (1:100, Eioscience); nd AF647 conjugted donkey nti-mouse IgG (1:500, Jckson ImmunoReserch Ls, West Grove, PA, USA). For this stin, the secondry ntiodies used were donkey nti-rit AF488 (1:200) nd donkey nti-rt AF594 (1:200) (Jckson ImmunoReserch). The second stin pnel used oth rit nti-iba-1 nd rt nti-mc2 (oth t 1:100, Eioscience), with nti-rit AF488 nd nti-rt AF594 s secondry ntiodies (Jckson ImmunoReserch). A third stin pnel contined rt nti-cd4 (1:100, Eioscience); rit nti-b220 (1:100, Affymetrix); nd got nti-c3 (1:100, MP Biomedicls). For this stin, the secondry ntiodies used were donkey nti-rt AF 594, donkey nti-rit AF488, nd donkey nti-got AF647 (ll 1:100, Jckson ImmunoReserch). Finlly, the fourth stin pnel contined rt nti-fironectin (1:100, Acm); got ntilumin (1:100, Bethyl Lortories); nd AF647 conjugted donkey nti-mouse IgG (1:500, Jckson Immuno Reserch). For this stin, the secondry ntiodies were donkey ntirt AF488 nd donkey nti-got AF594 (1:100, Jckson ImmunoReserch). To stin for neutrophils, we stined skin nd rin sections with rt nti-ly6g (1:100, BD- Bioscience), nd used donkey nti-rt AF488 (1:100, Jckson ImmunoReserch) s the secondry ntiody. For ssessment of poptotic cells in prffin tissue, the In Situ Cell Deth Detection Kit, Fluorescein (Sigm) ws used ccording to the mnufcturer s instructions. Behviorl testing MRL/lpr mice spontneously disply neuropsychitric phenotype, including cognitive normlities (memory deficits) nd depression-like ehvior. To ssess the effects of BI-BTK-1 on rin disese, neuroehviorl testing ws performed s previously descried [18]. Briefly, in the oject plcement test mice were exposed to two identicl ojects in different loctions within n ren for 5 minutes. The mice were then removed, nd fter 25-minute retention intervl returned to the testing ren, where one of the ojects hd een moved to novel loction. The reltive time the mouse spent exploring the ojects ws mesured nd the percent preference clculted. In the oject recognition test, the mice were put into n ren nd llowed to explore two identicl ojects for 3 minutes. Mice were then removed, sujected to 90-minute retention period nd returned to the ren, where one of the ojects hd een replced with novel oject. The reltive time spent with the ojects ws mesured, nd the percent preference ws clculted. Cognitively norml mice (i.e. with intct memory) will exhiit preference for ojects in new loction (oject plcement test) or novel oject (oject recognition test) [18]. To mesure depression-like ehvior, the Porsolt swim test ws used s previously descried [18]. Briefly, mice were plced into tnk of wter t room temperture. After n djustment period of one minute, the mount of time spent immoile ws mesured s n indiction of despir nd depression-like ehvior. RT-PCR Snp-frozen skin ws homogenized in Trizol using Retsch MM300 Tissue Lyser to collect RNA. Chloroform ws then dded nd the queous phse ws collected nd processed using the Agencourt RNAdvnced tissue kit tht ws modified for utomtion on Biomek FXp from Beckmn. RNA ws quntified on Nno- Drop 8000 instrument nd RNA qulity ws ssessed sed on RNA integrity numers using the Agilent 2200 Tpe Sttion. Reverse trnscription ws chieved using the TqMn Reverse Trnscription Regents Kit (Applied Biosystems). The resultnt cdna ws used in ViiA 7 Rel-Time PCR system (Applied Biosystems) using mouse-specific proes from Applied Biosystems. Cytokine protein expression Protein ws isolted from the skin using Tper uffer (Thermo Fisher Scientific, Wlthm, MA, USA), nd the isoltedproteinthenusedinbiolegend slegendplexmouse Inflmmtion Pnel (13-plex) per the mnufcturer s instructions. Flow cytometry Brins were hrvested from mice perfused with ice cold PBS, nd then the choroid plexus of the third, fourth, nd lterl ventricles ws dissected out nd comined. Choroid plexuses were infused with digestion uffer (2.5 mg/ml Lierse TL (Roche) nd 1 mg/ml of DNse I (Roche) in Hnk s lnced slt solution (HBSS) plus mgnesium nd clcium), cut into smll pieces nd put into C-tues
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 Pge 4 of 11 (Miltenyi). C-tues were positioned on MACS dissocitor nd run on the m_rin_3 protocol, fter which they were plced in n incutor for 30 minutes t 37 C with shking t 200 rpm. After incution, C-tues were positioned ck on the MACS dissocitor nd run on the m_rin_3 protocol. The relesed cells were then pssed through 40-μm nylon filter with cell msher, nd filters were wshed with 50 ml of wsh uffer (1% BSA in HBSS plus mgnesium nd clcium). Cells were stined with Fixle Viility Dye efluor 506 (ebioscience), incuted with Fc-Block (BD Bioscience) nd stined with the pproprite fluorochromeconjugted ntiodies (Additionl file 1: Tle S1). Cell counts were determined using 123count ebeds Counting Beds ccording to the mnufcturer s instructions (ebioscience). Dt were cquired on BD LSR II flow cytometer (BD Biosciences, Sn Jose, CA, USA). Compenstion nd nlysis of the flow cytometric dt were performed using Flowjo softwre (TreeStr, Ashlnd, OR, USA). Fluorescence minus one controls were used when necessry to set up gtes. The gting strtegy is illustrted in Additionl file 2: Figure S1. Quntittion of circulting IgG Serum IgG levels were mesured y ELISA, s previously descried [8]. Sttistics nd dt nlysis Dt were nlyzed using Grphpd Prism with the pproprite sttisticl tests. Grphs were prepred using the sme softwre. P vlues 0.05 were considered significnt. Results BI-BTK-1 tretment prevents mcroscopic skin pthology in MRL/lpr mice MRL/lpr mice were treted with control chow or chow formulted with the BTK inhiitor, BI-BTK-1, strting t 8 9 weeks of ge until the time of scrifice (~25 weeks of ge). BI-BTK-1 tretment significntly meliorted the skin lesions seen in control mice y 19 weeks of ge (Fig. 1). Furthermore, this protection ws mintined until the time of scrifice, t which point only 5/12 (42%) of the BI-BTK-1 treted mice hd ny signs of skin disese, wheres 11/12 (92%) of the control mice hd visile cutneous involvement (p < 0.0001) (Fig. 1). While some BI-BTK-1 treted mice still displyed lopeci or minor erythem, the skin ppered significntly helthier thn in the control-treted counterprts (Fig. 1, c). In contrst, control-treted mice developed severe mcroscopic lesions chrcterized y lopeci, erythem, scling, nd thickening of the skin on oth the fce nd dorsl thorx (Fig. 1c). BI-BTK-1 tretment significntly improves skin histopthology Control-treted MRL/lpr mice displyed histopthologic fetures of CLE, including thickening of the epidermis (hyperkertosis) nd cellulr infiltrtion (Fig. 2). In ddition to lleviting mcroscopic lesions, we found tht tretment with BI-BTK-1 significntly improved cutneous histopthology compred to control MRL/lpr mice (Fig. 2). Evlution of the lindly scored sections confirmed tht BI-BTK- 1 treted mice hd significntly improved skin rchitecture compred to control mice (Fig. 2c). c Fig. 1 Cutneous lesions in MRL/lpr mice. Mcroscopic lesions were scored over the course of the experiment up until the time of scrifice (). c BI-BTK-treted mice hd meliorted mcroscopic skin lesions s compred to control-treted mice. Three representtive mice re shown from ech group. Shown re the results from one experiment (BI-BTK-1, n = 12; control, n = 12) (**p < 0.01, ****p < 0.0001)
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 Pge 5 of 11 c Fig. 2 Skin histology. Control-treted MRL/lpr mice t 26 weeks of ge disply severe inflmmtory skin disese, s mrked y cellulr infiltrtion (smll rrows) nd hyperkerotosis (strs), which is mrkedly improved in BI-BTK-1-treted mice. Representtive imges re tken t 10 nd show mice in the treted nd control groups. The sections were lindly ssessed nd ssigned score. c BI-BTK-1, n = 12; control, n =9(*p < 0.05) BI-BTK-1 tretment prevents immune cell ccumultion in the skin To further chrcterize the effects of BTK inhiition on spontneous skin lesions in MRL/lpr mice, sections were stined for commonly infiltrting cells in CLE, nmely mcrophges (IBA-1 + ) nd T cells (CD4 + ), to ssess the effect of BTK inhiition on immune cell infiltrtion. Additionlly, sections were stined for IgG to ssess the effect of BI-BTK-1 on immunogloulin deposition in the skin. While we found no significnt difference in infiltrtion of B cells etween the groups (dt not shown), skin from BI-BTK-1 treted mice exhiited distinctly fewer mcrophges, T cells, nd IgG deposition compred to control mice (Fig. 3 nd ). We lso stined for neutrophils in the skin, ut found no significnt difference etween the BI-BTK-1-treted nd control-treted groups (dt not shown). The intensity of ech stin ws mesured using ImgeJ, verifying tht BI-BTK-1-treted mice hd significntly diminished infiltrtion of oth IBA-1+ mcrophges nd CD4+ T cells, nd ttenuted IgG deposition (Fig. 3c). Circulting serum IgG levels were lso significntly decresed with BI-BTK-1 tretment (Fig. 3c, right pnel). To further chrcterize the skin-infiltrting mcrophges, sections were lso stined with Mc-2, mcrophge ctivtion mrker. As cn e seen in Fig. 3d, not only were there fr lrger numers of IBA-1+ mcrophges in the lesionl skin of control mice, lrge mjority of them were Mc-2+ s well. BI-BTK-1 modultes expression of inflmmtory meditors in the skin We ssessed the reltive expression of mrna for vrious inflmmtory meditors in the skin. As seen in Fig. 4, control-treted MRL/lpr mice hd incresed TNF, monocyte chemottrct protein 1 (MCP-1), IL-10, IL-27, nd grnulocyte mcrophge-colony stimulting fctor (GM-CSF) s ssessed y RT-PCR. However, tretment with BI-BTK-1 significntly reduced the expression of these inflmmtory cytokines (Fig. 4). We lso ssessed the levels of vrious inflmmtory cytokines in the skin t the protein level. As seen in Fig. 4, BI-BTK-1 decresed the levels of vriety of cytokines previously ssocited with CLE pthogenesis [19], including TNF, IL-6, IL-17A, nd IL-10. Additionlly, the mcrophge relted cytokines MCP-1 nd GM- CSF were lso decresed y BI-BTK-1 tretment (Fig. 4). Interferon-γ (IFNγ) nd IL-1β levels, however, were not ffected y BTK inhiition (dt not shown). BI-BTK-1 tretment improves cognitive function in MRL/ lpr mice MRL/lpr mice disply cognitive dysfunction, including impired visul nd sptil memory, eginning round 16 weeks of ge [18]. We performed ehviorl testing on the BI-BTK-1 treted mice nd control mice t 17 weeks of ge, with mice sujected to oth the oject plcement (sptil memory) nd oject recognition (visul memory) tests. Treted mice hd significnt improvement in the
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 Pge 6 of 11 d c Fig. 3 Cellulr infiltrtion into the skin. Control-treted MRL/lpr mice disply prominent infiltrtion of the skin y mcrophges (green) nd T cells (red) (yellow rrows), nd IgG deposition (gry), which is significntly reduced in BI-BTK-1-treted mice. Representtive imges re tken t 10 nd show mice in the treted nd control groups. c The intensities of the mcrophge nd T cell stins (BI-BTK-1, n = 8; control, n = 9), nd the IgG stin (BI-BTK-1, n = 6; control, n = 6) were quntitted using ImgeJ. Right pnel, totl circulting IgG concentrtions were mesured in terminl serum (BI-BTK-1, n = 10; control, n = 11; p < 0.05). d Sections from control (top pnel) nd BI-BTK-1-treted (ottom pnel) mice were further co-stined with Mc-2 (red) nd IBA-1 (green) to ssess mcrophge ctivtion ( 20) (*p < 0.05, **p < 0.01, ***p < 0.001) oject plcement test, indicting preserved sptil memory (Fig. 5); treted mice hd men preference for explortion of the oject t the novel loction of 61 ± 13%, s compred to preference of 50 ± 15% in the control mice (p < 0.05). A trend towrd improvement ws lso oserved in the oject recognition test; however, this ws not sttisticlly significnt (Fig. 5). MRL/lpr mice exhiit depression-like ehvior s erly s 5 weeks of ge [18]. The effect of BI-BTK-1 tretment on this ehvior ws ssessed t 17 weeks of ge. We found tht strting tretment with BI-BTK-1 t 8 9 weeks of ge did not improve depression-like ehvior, s ssessed y the Porsolt forced swim test (Fig. 5c). Open field tests reveled tht neither mouse group hd git deficits or musculoskeletl wekness, s oth groups displyed similr totl trck lengths (dt not shown). There were no differences in center trck length, center time, or center visits, to indicte chnges relted to nxiety or risk-seeking ehvior (dt not shown). BI-BTK-1 tretment improves rin histologic ppernce We ssessed H&E-stined rin sections to determine the effect of BI-BTK-1 tretment on infiltrtion of inflmmtory cells into the centrl nervous system. Specificlly, we focused on the choroid plexus, the site of the lood-cererospinl-fluid rrier, where we nd others hve previously descried mrked lymphocyte infiltrtion in the MRL/lpr strin strting t 16 weeks of ge [20]. While controltreted MRL/lpr mice hve prominent cellulr infiltrtion into the choroid plexus in the region of dorsl fourth ventricle (Fig. 5d), BI-BTK-1-treted mice hd mrked diminution in the numer of infiltrting cells (Fig. 5e). When the sections were lindly scored for stroml expnsion nd the degree of infiltrting immune cells, we found tht BI-BTK-1 tretment significntly reduces choroid plexus pthology s compred to control mice (Fig. 5f). We lso evluted the presence of poptosis within the cortex, choroid plexus, nd hippocmpus, ut found no significnt differences in the numer of TUNELpositive cells etween BI-BTK-1-treted nd controltreted mice (dt not shown). Furthermore, there were no notle differences in mrkers of loodrin rrier permeility, s ssessed y stining for fironectin, lumin, nd IgG (dt not shown).
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 Pge 7 of 11 Fig. 4 Inflmmtory meditor expression in the skin. RT-PCR ws performed on mrna isolted from lesionl skin nd ssessed for the reltive expression of vrious inflmmtory cytokines. Protein lystes were prepred from lesionl skin, nd cytokine expression quntitted s descried in Methods.BI-BTK-1,n = 12; control, n =9(*p <0.05, **p < 0.01, ***p <0.001) BI-BTK-1 reduces ccumultion of T cells, B cells, nd mcrophges in the choroid plexus To further investigte if the improvement in the neuroehviorl phenotype in BI-BTK-1-treted mice is ssocited with decresed rin infiltrtion y prticulr cell types, flow cytometry ws performed on cells isolted from the choroid plexus of BI-BTK-1-treted nd control-treted MRL/lpr mice ge 17 18 weeks. We found tht BI-BTK-1-treted mice exhiited significntly decresed infiltrting leukocytes (CD45+ cells), including CD4+ T cells, CD8+ T cells, nd CD19+ B cells, s well s mcrophges nd monocytes (Fig. 6). To confirm the flow cytometric results in nlyzing whether BI-BTK-1 ws preventing the specific ccumultion of prticulr types of immune cells in the choroid plexus, rin sections were stined for mcrophges (IBA- 1), T cells (CD4), nd B cells (B220). We found tht BTK inhiition prevented the ccumultion of ll three cell types (Fig. 6, c). Furthermore, decrese in loclly deposited IgG nd C3 ws noted s well (Fig. 6, c). We ssessed the stte of mcrophge ctivtion in the ccumulting IBA-1+ cells y stining for Mc-2. As cn e seen in Fig. 6d, not only do the control mice hve more IBA-1+ mcrophges ut lrger percentge re Mc-2 positive, indicting more inflmmtory mcrophge phenotype ccumulting in the choroid plexus of control-treted mice. Finlly, no meningful ccumultion of neutrophils ws oserved in the choroid plexus of either control-treted or BI-BTK-1- treted mice (dt not shown). Discussion Neuropsychitric mnifesttions nd skin involvement re two common nd potentilly serious end-orgn pthologies ssocited with SLE, oth of which present pressing unmet therpeutic need. Current tretment still predominntly relies on rodly immunosuppressive medictions, which while effective in some ptients re ssocited with dngerous side effects nd no gurntee of remission. In the study presented here, we showed tht BI-BTK-1 tretment hd drmtic effects in preventing the development of cutneous lesions nd meliorting the cognitive dysfunction commonly seen in clssic lupus niml model. Whether or not BTK inhiition is etter thn existing tretments cnnot e stted with confidence t this time, since our study design did not include n ctive comprtor rm (e.g. tretment with high-dose steroids nd/or cyclophosphmide). Moreover, lthough well eyond scope of the present study, future experiments will need to investigte the effect of delyed tretment on more estlished disese phenotypes, nd on long-term survivl. Nevertheless, nrrowing the cells/pthwys trgeted in SLE is, t the very lest, likely to improve the therpeutic index. Considering the vrious cell types tht hve een implicted in the pthogenesis of SLE, therpeutic BTK inhiition would lrgely directly ffect B cells nd mcrophges, oth of which hve een implicted in skin nd rin disese. Although the precise mechnism underlying ech individul type of end-orgn pthology in lupus hs yet to e fully elucidted, utontiody deposition is thought to e importnt in oth types of tissue [5, 21]. Other potentil contriutions of B cells to the mechnisms of trget orgn injury in lupus re ntigen presenttion nd cytokine secretion [22]. Furthermore, we hve previously shown tht mcrophges re importnt for the development of oth spontneous nd ultrviolet B irrdition-induced skin lesions in lupus mice nd in the pthogenesis of murine NPSLE [7 9]. Other investigtors hve previously reported the use of vrious BTK inhiitors in lupus models, including NZB/W, MRL/lpr, SLE1,3, nd BXSB-Y strins nd nephrotoxic serum nephritis (the ltter nephritis-limited model inducile in non-utoimmune mice following pssive trnsfer of nephritogenic ntiodies). In ll cses, n improvement in kidney disese ws reported [15, 23 26]. Hutchenson et l. lso noted tht in ddition to the preserved kidney function in SLE1,3 mice treted with BTK inhiition there ws lso
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 d e Pge 8 of 11 c f Fig. 5 Neuroehviorl testing nd choroid plexus infiltrtion. BI-BTK-1-treted mice hve improved cognitive function s evidenced y significnt improvement in the oject plcement test (). A trend towrd improvement in the oject recognition test ws present s well (). Tretment hd no effect on depression-like ehvior (c) (BI-BTK-1, n = 12; control, n = 12). d MRL/lpr mice demonstrte mrked choroid plexus infiltrtion with lymphocytes nd mcrophges, s seen here in section of the fourth ventricle. Tretment with BI-BTK-1 meliortes this infiltrtion (e). Representtive imges re tken t 10 ( nd d) nd 20 (c) nd shown from mice in the treted nd control groups, nd lindly scored using the scle descried in Methods (f). BI-BTK-1, n = 12; control, n = 8 (**p < 0.01) c d Fig. 6 Chrcteriztion of the choroid plexus infiltrtes. Flow cytometric nlysis of choroid plexus (CP) infiltrting immune cells in smll pilot study (BI-BTK-1, n = 2; control, n = 2) reveled infiltrtion of leukocytes, T cells, B cells, nd mcrophges. Immunofluorescent stining confirmed lrge popultions of mcrophges (green) ccumulting in the choroid plexus of control mice (), nd incresed IgG deposits (mgent) compred to BI-BTK-1-treted mice (c). Control mice lso hd incresed numers of T cells (red), B cells (green), nd C3 complement deposition (gry). d Mcrophges infiltrting the control-treted mice CPs hd n ctivted phenotype (Mc-2+) compred to the BI-BTK-2-treted mice. Representtive imges re shown in ll pnels (BI-BTK-1, n = 7; control, n = 7)
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 Pge 9 of 11 decrese in utorective humorl immunity, with delyed production of utontiodies nd decresed splenomegly. In NZB/W mice, BTK inhiition downregulted the expression of interferon relted genes in the spleen, finding ttriuted to decresed systemic ctivtion of mcrophges vi their Fc receptors. These studies collectively show tht BTK inhiition in lupus models cn mitigte kidney disese. However, the effect of BTK inhiition on other centrl mnifesttions of murine (nd humn) lupus, including cutneous nd neuropsychitric disese, hs not een studied to dte. These effects of BTK inhiition in ntiodymedited nephritis [15], nd initil studies reporting good therpeutic results with resonle sfety in humn disese (for non-lupus indictions) [27], led us to the current study which ws to focus the investigtion on the effects of BTK inhiition in lupus-ssocited extr-renl involvement. Skin lesions in MRL/lpr were significntly improved y BI-BTK-1 tretment, oth mcroscopiclly nd histologiclly. Treted mice lso hd less cellulr infiltrtion into the skin, nd less IgG deposition; decresed numers of oth mcrophges nd T cells were seen, cell types which impct the lupus-ssocited cutneous disese vi effector functions nd cytokine relese. Further, the mcrophges seen in BI-BTK-1-treted mice hd less ctivted phenotype. Indeed, BI-BTK-1 tretment decresed the concentrtion of skin cytokines, including severl previously ssocited with CLE pthogenesis such s IL-6, IL-17, nd TNF. BTK-dependent signling is further known to promote mcrophge polriztion towrds n inflmmtory phenotype (M1) in vitro [13]. Consequently, decresed expression of these prticulr cytokines is likely to e medited through preventing BTK-medited mcrophge polriztion. Furthermore, BTK my lso e importnt for downstrem signling of TLRs, specificlly downstrem of TLR4-dependent expression of IL-10 [14, 28]. The relevnce of the ltter mechnism here is supported y the decrese we found in skin IL-10 levels in BTK-I-treted mice. Finlly, it is importnt to cknowledge tht the less inflmmtory cytokine environment present in treted mice my hve indirectly contriuted to ttenuted mcrophge ctivtion. Nevertheless, our previous oservtion tht BI- BTK-1 inhiits the secretion of IL-6, TNF, nd IL-1 from isolted immune-complex-stimulted monocytes [15] is supportive of conclusion tht the reduced mcrophge inhiition we oserved here in vivo is t lest prtly due to the contriution of direct inhiitory effect of BI- BTK-1 tretment. We found tht BI-BTK-1 tretment inhiited mcrophge recruitment to inflmed skin. Studies in monocytes isolted from ptients with mutted BTK hve reveled tht BTK defective monocytes hve decresed chemotctic response [29]. Further, we found here tht BI-BTK-1 tretment lso decresed MCP-1 nd GM- CSF, oth of which re importnt for mcrophge recruitment nd survivl. Becuse mcrophges cn potentilly secrete lrge mounts of cytokines, especilly once ctivted y immunogloulin deposited t the dermoepiderml junction in CLE [1], decresed mcrophge ccumultion is likely direct eneficil result of BI- BTK-1 tretment. BTK inhiition would lso prevent mcrophge ctivtion vi oth Fc receptors (s we hve previously shown [15]) nd possily through TLRs, oth of which would e ctivted y utontiodies or nucler ntigens deposited in the skin. Thus, the effect of BI-BTK-1 on mcrophges could potentilly e mjor mechnism y which this drug meliortes skin disese. The decrese in mcrophge ctivtion, nd consequentil decrese in inflmmtory cytokine nd chemokine expression, my contriute together to the decresed cellulr infiltrtion seen in the skin, oth for mcrophges nd T cells (even though the ltter cell type is not directly impcted y BTK inhiition). Interestingly, Rnkin [25] nd Hutcheson [23] lso found tht BTK inhiition in lupus cn ffect the T cell comprtment, nd suggested tht B cells re required for T cell mintennce [25]. In future studies, it would e interesting to isolte primry mcrophges from BI-BTK-1-treted mice to crefully ssess their phenotypes ex vivo nd responses to vrious inflmmtory stimuli. We nd others hve previously demonstrted multiple effects y which BTK inhiition cn modulte B cell function in lupus models. To riefly summrize some of the relevnt effects, inhiition of BTK in vitro prevents B cell ctivtion in response to BCR-medited cross linking, nd interferes with specific intrcellulr signling pthwys nd nti-cd40-stimulted prolifertion [24, 25, 30, 31]. In vivo, BTK inhiited spontneous germinl center formtion in lupus mice, nd decresed splenic B cell numers (mrginl zone, folliculr, B1, plsmlsts, nd plsm cells), nd circulting B cells, ntiodies nd/or utontiodies [23 25, 31]. The mgnitude of the effect oserved nd which B cell suset, ntiody isotype, or specificity ws most ffected ws t times vrile etween these studies, depending on the niml model used, drug dministrtion protocol, nd of course the ctul molecule used for BTK inhiition. Nevertheless, it is cler tht BTK tretment in lupus models is consistently ssocited with mrked effect on multiple B cell comprtments nd functions. Therefore, it is highly likely tht the eneficil effects of BI-BTK-1 on skin nd rin disese were rought out through its direct effects on B cell specific ctions. Although it is possile tht some B cell modultory effects re more importnt thn others, the significnt enefit provided y BTK inhiition in lupus niml models is proly function of multiple dditive nd/or synergistic effects on this key cell type.
Chlmers et l. Arthritis Reserch & Therpy (2018) 20:10 Pge 10 of 11 In ddition to the improvement in cutneous disese, we found tht BI-BTK-1 tretment improved NPSLE mnifesttions. Specificlly, treted mice hd improved cognitive function. Although the oserved deficits in memory were not totlly reversed, perhps longer tretment would e required to demonstrte more complete response. In this regrd, the decresed ccumultion of mcrophges, T cells, nd B cells in the choroid plexus ws highly significnt nd very encourging. BTK inhiition my inhiit lymphoid chemotxis through the CCL20/CCR6 pthwy, s oth B cells nd T cells re CCR6+, nd expression of CCL20 is higher in the choroid plexus thn elsewhere in the rin not only during experimentl llergic encephlomyelitis ut in norml mice s well [32]. BTK hs lso een shown to e importnt in neutrophil recruitment nd function [33, 34]; in model of sterile inflmmtion induced y focl heptic necrosis, BTK deficiency decresed neutrophil recruitment to the loction of injury [33]. In our study, however, we found no evidence of neutrophil infiltrtion into the choroid plexus, nd in the skin we sw no differences in neutrophil numers etween the control nd treted mice. These findings re consistent with the reported heterogeneity in the role of BTK in neutrophil physiology [35]. The mcrophges in the rin, known s microgli, re mjor source of inflmmtory cytokines which re lso elieved to e importnt in NPSLE pthogenesis [8]. Similr to wht we discussed regrding the skin, BI- BTK-1 inhiition my hve polrized the microgli (nd/ or rin infiltrting mcrophges) to n M2-like phenotype, even in the presence of M1 driving stimuli. In the choroid plexus, decresed Mc-2 stining of IBA-1+ mcrophges indictes less ctivtion of these cells. Additionlly, utontiodies re lso thought to contriute to NPSLE. BTK inhiition nd its effect on B cells could potentilly hve ffected this s well. Overll, BTK inhiition presents itself s potentilly vlule therpeutic option for NPSLE, in ddition to ny slutry effects on the skin disese. Conclusions BTK inhiitors (e.g. irutini) re lredy eing used in humn disese for hemtologic indictions (chronic lymphocytic leukemi nd smll lymphocytic lymphom), highlighting the rel trnsltionl potentil of this pthwy [27]. Irutini is reltively well-tolerted in humns, with side effects including upper respirtory trct infection, ftigue, nd dirrhe, lthough none so severe to require its discontinution [27]. BI-BTK-1 ws designed to hve improved selectivity nd potency over existing inhiitors [15], potentilly minimizing the side effects currently seen. Overll, we elieve BI-BTK-1 my hold significnt promise for the tretment of resistnt SLE mnifesttions, including rin nd skin disese. Additionl file Additionl file 1: Tle S1. Antiodies utilized for choroid plexus flow cytometric nlysis. (DOCX 12 k) Additionl file 2: Figure S1. Flow cytometry gting strtegy. Corticl nd choroid plexus tissue from MRL/lpr mice treted or not with BI-BTK-1 were nlyzed y flow cytometric nlysis. Red rrows denote sequentil gted popultion (red oxes). Blck rrows denote sequentil non-gted popultion. (DOCX 358 k) Arevitions BCR: B cell receptor; BSA: Bovine serum lumin; BTK: Burton s tyrosine kinse; CLE: Cutneous lupus erythemtosus; ELISA: Enzyme-linked immunosorent ssy; H&E: Hemtoxylin nd eosin; IL: Interleukin; MCP- 1: Monocyte chemottrct protein 1; NPSLE: Neuropsychitric lupus; PBS: Phosphte-uffered sline; SLE: Systemic lupus erythemtosus; TLR: Tolllike receptor; TNF: Tumor necrosis fctor Acknowledgements We thnk Elizeth Glynn (Boehringer Ingelheim) for technicl help in coordinting the studies. Funding Boehringer Ingelheim (Ridgefield, CT) funded these studies. With the exception of the uthor contriutions detiled in the section elow, the compny did not hve role in the design of the studies, collection/ nlysis/interprettion of the dt, or writing of the mnuscript. Avilility of dt nd mterils The dtsets used nd/or nlyzed during the current study re ville from the corresponding uthor on resonle request. Authors contriutions The study ws conceived nd designed y MR nd CP. BI-BTK-1 ws designed y TB. In vivo studies, neuroehviorl studies, skin scoring, nd IF stining were performed y SAC, JW, JD, nd AS. Flow cytometric experiments nd nlysis were performed y CMC, HMM, nd HP. RT-PCR ws performed y DW. The study ws overseen y GN, JSF, EK, MR, nd CP. The mnuscript ws written y SAC, MR, nd CP. All uthors reviewed nd pproved the sumission. Ethics pprovl Animl studies were pproved y the institutionl niml cre committee of the Alert Einstein College of Medicine (Bronx, NY, USA). Consent for puliction Not pplicle. Competing interests Todd Bosnc, Deorh We, Gerld Nozny, Jy Fine, Elliott Klein, nd Meer Rmnujm re full-time employees of Boehringer Ingelheim (Ridgefield, CT). The other uthors declre tht they hve no competing interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions. Author detils 1 Deprtment of Microiology nd Immunology, Alert Einstein College of Medicine, Bronx, NY, USA. 2 Division of Rheumtology, Northwestern University Feinerg School of Medicine, Chicgo, IL, USA. 3 Smll Molecule Discovery Reserch, Boehringer Ingelheim Phrmceuticls, Ridgefield, CT, USA. 4 Immunology nd Respirtory Disese Reserch, Boehringer Ingelheim Phrmceuticls, Ridgefield, CT, USA. 5 Division of Rheumtology, Alert Einstein College of Medicine, F701N, 1300 Morris Prk Ave, Bronx, NY 10461, USA.
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