Small Molecule Inhibition of Rab7 Impairs B Cell Class Switching and Plasma Cell Survival To Dampen the Autoantibody Response in Murine Lupus

Tonika Lam, Dennis V. Kulp, Rui Wang, Zheng Lou, Julia Taylor, Carlos E. Rivera, Hui Yan, Qi Zhang, Zhonghua Wang, Hong Zan, Dmitri N. Ivanov, Guangming Zhong, Paolo Casali and Zhenming Xu
J Immunol November 15, 2016, 197 (10) 3792-3805; DOI: https://doi.org/10.4049/jimmunol.1601427

Abstract

IgG autoantibodies mediate pathology in systemic lupus patients and lupus-prone mice. In this study, we showed that the class-switched IgG autoantibody response in MRL/Faslpr/lpr and C57/Sle1Sle2Sle2 mice was blocked by the CID 1067700 compound, which specifically targeted Ras-related in brain 7 (Rab7), an endosome-localized small GTPase that was upregulated in activated human and mouse lupus B cells, leading to prevention of disease development and extension of lifespan. These were associated with decreased IgG-expressing B cells and plasma cells, but unchanged numbers and functions of myeloid cells and T cells. The Rab7 inhibitor suppressed T cell–dependent and T cell–independent Ab responses, but it did not affect T cell–mediated clearance of Chlamydia infection, consistent with a B cell–specific role of Rab7. Indeed, B cells and plasma cells were inherently sensitive to Rab7 gene knockout or Rab7 activity inhibition in class switching and survival, respectively, whereas proliferation/survival of B cells and generation of plasma cells were not affected. Impairment of NF-κB activation upon Rab7 inhibition, together with the rescue of B cell class switching and plasma cell survival by enforced NF-κB activation, indicated that Rab7 mediates these processes by promoting NF-κB activation, likely through signal transduction on intracellular membrane structures. Thus, a single Rab7-inhibiting small molecule can target two stages of B cell differentiation to dampen the pathogenic autoantibody response in lupus.

This article is featured in In This Issue, p.3749

Introduction

Class switch DNA recombination (CSR) in the IgH locus substitutes the IgH constant (CH) region, such as Cμ of IgM expressed in naive B cells, with Cγ, Cα, or Cε, thereby giving rise to IgG, IgA, or IgE Abs. Together with somatic hypermutation (SHM), CSR is central to the maturation of the Ab response to pathogens, as class-switched Abs display wide tissue distributions and possess diverse biological effector functions (1). These traits also make IgG and IgA autoantibodies highly pathogenic and capable of mediating multiple organ damage in systemic lupus erythematosus (SLE) (2, 3). Unswitched IgM autoantibodies are mostly nonpathogenic and, rather, may mediate frontline immune protection as natural polyreactive Abs (4). Class-switched B cells further differentiate into memory B cells or plasma cells, which secrete Abs at a high rate. A hallmark of active lupus is the high number of IgG+ Ab-secreting cells (ASCs) that produce autoantibodies. These are diverse but generally target nuclear Ags, such as DNA (5, 6), making the mechanisms underlying B cell class switching and plasma cell generation/maintenance targets of new lupus therapeutics.

Similar to SHM, CSR requires activation-induced cytidine deaminase (AID, encoded by AICDA in humans and Aicda in mice). AID expression is mainly restricted in peripheral B cells activated by CD154 engagement of CD40 on the B cell surface or by complex Ags that engage both a TLR and the BCR (7). AID is elevated in B cells of lupus patients and lupus mice, consistent with the heightened CSR/SHM in these B cells (8), and AID deficiency abrogates IgG autoantibodies in lupus-prone MRL/Faslpr/lpr mice (8, 9). Inhibitors of AID deaminase activity are yet to be developed, thereby emphasizing the need for molecules that target the mechanisms underlying AID induction to dampen the class-switched pathogenic autoantibody response.

Ras-related in brain 7 (Rab7; encoded by RAB7A in humans and Rab7 in mice) is a small GTPase that, when bound to its GTP substrate, promotes endosome maturation and autophagy. As we have shown (10), Rab7 is induced in activated B cells in vivo (i.e., in peanut agglutinin [PNA]hi germinal center B cells) and in vitro, for example, by CD154 and TLR ligands, the same stimuli that induce AID expression and CSR. It plays a B cell–intrinsic role in Ab responses, as mice that conditionally knockout Rab7 in activated B cells cannot mount mature Ab responses to T cell–dependent or –independent Ags (10). Rab7 promotes CSR (to IgG, IgE, and IgA) and does so by mediating AID induction, as enforced expression of AID rescues CSR in Rab7 knockout B cells. Furthermore, Rab7 plays an important role in CD40- or TLR-triggered activation of NF-κB, which directly regulates Aicda gene transcription by binding to the promoter and enhancers of this gene (1, 11, 12). Rab7 is, however, dispensable for Erk1/Erk2 activation and expression of Blimp-1, both of which critically mediate plasma cell generation (13, 14), and, as a consequence, B cell differentiation into plasma cells, suggesting that Rab7 and its associated intracellular membrane structures (i.e., endosomes) specify receptor-triggered signaling for selective gene expression and B cell differentiation processes (15). Whether Rab7 plays a role in the maintenance of plasma cells remains unclear.

In this study, we hypothesized that the lupus autoantibody response can be suppressed by inhibition of CSR in B cells and impairment of generation or maintenance of plasma cells, ideally by a single molecule that can target both cell types. To test this hypothesis, we have used a high-affinity and specific Rab7 inhibitor, CID 1067700. This has been identified by high-throughput screening as the only compound to affect the binding of purified recombinant Rab7 to GTP and GDP (16). By analyzing the level of activated Rab7 form (GTP-bound Rab7, Rab7-GTP) in B cells treated with CID 1067700 and using B cell–specific Rab7 knockout mice as well as retroviruses that enforced specific gene expression, we have verified the specific targeting of Rab7 by this small molecule in B cells and the consequent impairment in NF-κB activation. By using our defined in vitro B cell and plasma cell culture systems, we have further analyzed the impact of Rab7 inhibition on B cell class switching and plasma cell generation/survival as well as the role of Rab7-dependent NF-κB activation in these processes. Finally, by analyzing, to our knowledge for the first time, the in vivo effect of the Rab7 inhibition on Ab and autoantibody responses in normal C57BL/6 (C57) mice and two widely used lupus mouse strains, female MRL/Faslpr/lpr and C57/Sle1Sle2Sle3 mice (17, 18), we have outlined the potential of Rab7 as a therapeutic target in lupus and possibly other NF-κB–, CSR-, and/or plasma cell–mediated disease conditions.

Materials and Methods

Mice, drug treatment, immunization, and infection

Female mice were used in all experiments. C57 (stock no. 000664), MRL/Faslpr/lpr (stock no. 000485), and C57/Sle1Sle2Sle3 (stock no. 007228) mice were from The Jackson Laboratory and maintained in a pathogen-free vivarium. Conditional Rab7 knockout Igh+/Cγ1-creRab7fl/fl mice and their wild-type Igh+/Cγ1-creRab7+/fl littermates on the C57 background were as described (10). Rosa26+/fl-STOP-fl-Ikkβca mice on the C57 background (19) were from The Jackson Laboratory (stock no. 008242).

For in vivo treatment, CID 1067700 (MLS000673908, SID 24798006, SID 57578339; Glixx Laboratories) dissolved in DMSO (“nil;” stock concentration 40 mM, 16 mg/ml) was diluted with the solvent to the final volume of 50 μl and injected i.p. once per week at the dose of 16 mg/kg body weight. This dose was well tolerated by adult mice at all ages (data not shown); it is within the dose range used in patients and animal models for several drugs with comparable EC50 (10–100 nM) to their respective targets (higher doses of CID 1067700 led to variable mouse death, perhaps due to the putative off-targeting effect of the drug). C57, MRL/Faslpr/lpr, and C57/Sle1Sle2Sle3 mice injected i.p. with nil (50 μl) showed no difference in examined parameters, as compared with their respective counterparts without any injection (data not shown). For survival studies and skin lesion analyses, MRL/Faslpr/lpr mice were treated with nil or CID 1067700 for 10 wk and maintained until moribund (e.g., showing signs of severe loss of mobility, hunched back, piloerection, ruffled fur, dyspnea, gasping, and weight loss), at which point they were euthanized. For other clinical, serological, cellular, and molecular analyses (see below), mice were treated for 7 wk in a double-blind manner, as their identities were made unknown to investigators who performed these assays.

For immunization, C57 mice were treated with nil or CID 1067700 7 d before i.p. injection with 100 μg of (4-hydroxy-3-nitrophenyl)acetyl (NP)–chicken γ-globulin (CGG) (in average 16 molecules of NP conjugated to 1 molecule of CGG; Biosearch Technologies) in 100 μl of aluminum hydroxide (Imject Alum; Pierce) or 25 μg of NP-LPS (0.2 NP molecule conjugated to 1 LPS molecule; Biosearch Technologies) in 100 μl of PBS. Mice were treated with nil or CID 1067700 once every 3 d until being sacrificed.

For Chlamydia muridarum infection, the Nigg strain was propagated in HeLa cells for the isolation of the Nigg3G0.10.1 clone, as previously described (20). Purified Nigg3G0.10.1 elementary bodies were used to infect 6-wk-old C57 mice intravaginally with 2 × 105 inclusion-forming units (IFUs). Mice were injected with 2.5 mg of medroxyprogesterone (Depo-Provera; Pharmacia and Upjohn) s.c. at day –5, to increase the susceptibility, and with nil or CID 1067700, starting at day –7, once a week for 5 wk. Mice were monitored for vaginal live organism shedding up to day 63. All protocols were in accordance with rules and regulations of the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio.

Human and mouse primary B cells

Human PBMCs were prepared, following a standard protocol, from peripheral blood of de-identified SLE patients and healthy subjects, as collected by venipuncture. SLE patients, who were recruited with informed consent under the protocol approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio, fulfilled at least four 1982 American Rheumatism Association revised criteria for SLE and had the SLE disease activity index >3. All donors were free of infection by HBV, HCV, human papillomavirus, HIV, and EBV. To prepare human B cells for stimulation, PBMCs (5 × 107) were subjected to negative selection (against CD2, CD14, CD16, CD27, CD36, CD43, and CD235a) using human naive B cell isolation kit II (Miltenyi Biotec) following the manufacturer’s instructions, resulting in >95% IgD+ B cells (generally >3 × 106). Cells were analyzed or seeded at 3 × 105 cell/ml for culturing in RPMI 1640 medium (Invitrogen) supplemented with FBS (10% v/v; HyClone Laboratories), penicillin-streptomycin/amphotericin B (1% v/v), and 50 μM 2-ME (RPMI-FBS).

Single-cell suspensions were prepared from the spleen and pooled axillary, inguinal, and cervical lymph nodes. Lymph node cells were directly lysed for immunoblotting studies. Spleen cells were resuspended in ACK lysis buffer (Lonza) to lyse RBCs and, after quenching with RPMI-FBS, were resuspended in PBS for analysis or further preparation. To isolate B cells, spleen cells were subjected to negative selection (against CD43, CD4, CD8, CD11b, CD49b, CD90.2, Gr-1, or Ter-119) using an EasySep mouse B cell isolation kit (Stemcell Technologies) following the manufacturer’s instructions, resulting in preparations of >98% B cells. After pelleting, B cells were resuspended in RPMI-FBS before further analysis or stimulation. For cell sorting, spleen cells were stained with fluorophore-labeled anti-CD19 mAb, anti-CD138 mAb, anti-TCR mAb, and/or PNA to purify B cells, activated B cells, and plasma cells, as indicated, resulting in preparations that were at least 98% pure by postsorting analysis. Bone marrow cells were isolated form tibia and fibula for myeloid cell differentiation and ELISPOT experiments.

B cell culture, stimulation, and drug treatment

Human B cells were stimulated with CD154 (3 U/ml) in the presence of recombinant IL-4 (20 ng/ml; BioLegend) and IL-21 (50 ng/ml; R&D Systems) for 48 h for trancript analyses or 120 h for flow cytometry analysis of class-switched IgG1 and other markers. CD154 was prepared as membrane fragments isolated from Sf21 insect cells infected by CD154-expressing baculovirus, as we described (10, 21); membrane fragments from noninfected Sf21 cells failed to stimulate B cells. For CSR induction in mouse B cells, spleen B cells that were cultured (3 × 105 cell/ml) in RPMI-FBS were stimulated for 96 h (or 48 h for transcript and protein analyses) by the following primary stimuli: CD154 (3 U/ml), LPS (3 μg/ml, from Escherichia coli, serotype 055:B5, deproteinized by chloroform extraction; Sigma-Aldrich), or R-848 (30 ng/ml; InvivoGen), a ligand of TLR7, which promotes lupus autoantibody responses in a B cell–intrinsic manner. rIL-4 (4 ng/ml), for CSR to IgG1 and IgE, and TGF-β (2 ng/ml; R&D Systems), for CSR to IgA, and anti-Igδ mAb (clone 11-26c conjugated to dextran, anti-δ/dex, 100 ng/ml; Fina Biosolutions), which crosslinks IgD BCR to potentiate CSR to IgA in primary B cells, were added as indicated. To induce CSR to IgA in the mouse IgM+ CH12F3 lymphoma cell line, these B cells (105 cell/ml) were cultured in RPMI-FBS and stimulated with CD154 (3 U/ml) or, to our knowledge as first shown here, by LPS at low concentrations (100 ng/ml), plus IL-4 and TGF-β for 48 h. To generate plasma cells in vitro, purified B cells were stimulated with LPS plus IL-4, TGF-β, anti-Igδ mAb conjugated to dextran (anti-δ/dex), and retinoic acid (RA, 10 μM; Sigma-Aldrich) for 66 h. This condition, as we first found here, resulted in CD19loCD138hi plasma cells representing >70% of live cells in the culture.

To treat human and mouse B cells in vitro with the Rab7 inhibitor, CID 1067700 was diluted in DMSO and added to cell cultures to the final concentration of 40 μM (or as indicated). CID 1067700 or nil was added either at the time when B cell stimulation started, or 66 h after B cells were stimulated with LPS plus IL-4, TGF-β, anti-δ/dex, and RA, as indicated, for analysis of plasma cell survival (see below).

Analysis of CSR, cell proliferation, and survival

To analyze IgG-expressing B cells and plasma cells in vivo, spleen cells (2 × 106) were first stained with fluorophore-labeled mAbs to surface markers and 7-aminoactinomycin D (7-AAD) (Supplemental Table I). After washing, cells were resuspended in the BD Cytofix/Cytoperm buffer (250 μl; BD Biosciences) and incubated at 4°C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were stained in the same buffer with fluorophore-labeled mAb to IgM, IgG1, IgG2a, or IgG2b followed by washing for flow cytometry analysis. Dead (7-AAD+) cells were excluded from analysis. For CSR analysis of B cells stimulated in vitro, B cells were analyzed by flow cytometry for surface expression of IgG and IgA as well as intracellular expression of IgE, as we have described (10, 21).

For B cell proliferation analysis in vivo, mice were injected i.p. with 2 mg of BrdU in 200 μl of PBS twice, with the first and second injection at 24 and 20 h prior to sacrificing, respectively. Spleen B cells were analyzed for BrdU incorporation (by anti-BrdU mAb staining) and DNA contents (by 7-AAD staining after cells were permeablized) using a BrdU flow kit (BD Biosciences) following the manufacturer’s instructions. For B cell proliferation analysis in vitro, naive B cells were labeled with CFSE (Invitrogen) following the manufacturer’s instructions and then cultured for 96 h in the presence of appropriate stimuli. Cells were analyzed by flow cytometry for CFSE intensity, which was reduced by half after completion of each cell division, as CFSE-labeled cell constituents were equally distributed between daughter cells. The number of B cell divisions was determined by the “proliferation platform” of FlowJo software.

To analyze B cell and plasma cell survival in MRL/Faslpr/lpr mice treated with CID 1067700 or nil in vivo, spleen cells were stained with anti-CD19 mAb, anti-CD138 mAb, anti-TCR mAb (to identify TCR−CD19+CD138− B cells and TCR−CD19−/loCD138+ plasma cells) in the presence of mAb clone 2.4G2, which blocks the FcγIII and FcγII receptors, and 7-AAD without permeabilization (to identify apoptotic/necrotic cells) and analyzed by flow cytometry. To analyze in vitro survival of immune cells isolated from MRL/Faslpr/lpr mice, spleen cells were cultured in RPMI-FBS for 24 or 48 h in the presence of CID 1067700 or nil. After staining with surface markers of different cell types and 7-AAD, cells were analyzed by flow cytometry. To analyze survival of in vitro–generated plasma cells (after B cell stimulation with LPS plus IL-4, IL-5, TGF-β, anti-δ/dex, and RA for 66 h), cells were washed with RPMI-FBS twice and cultured in RPMI-FBS, without any additional stimuli to block further plasma cell generation, for 24, 48, and 72 h, in the presence of nil or CID 1067700 (40 μM). Cells were collected and stained with mAbs to CD138 and CD19 (to mark plasma cells and B cells) as well as with annexin V and 7-AAD to distinguish live cells (annexin V−7-AAD−) from cells undergoing early (annexin V+7-AAD−) and late (annexin V+7-AAD+) apoptosis.

Retrovirus transduction

Retroviral vectors pMIG and pMIG-AID (encoding AID) were as described (10); pMIG-Cre (encoding the Cre recombinase) was from Addgene; pMIG-Rab7 and pMIG-Rab2a (encoding Rab7 and Rab2a, respectively) were constructed using specific oligonucleotides (Supplemental Table II). These vectors were cotransfected with the packaging plasmid into 293T cells to produce retroviruses, as described (10, 22). For transduction, B cells isolated from C57 or Rosa26+/fl-STOP-fl-Ikkβca mice were stimulated with LPS for 24 h in the presence of nil or CID 1067700, as indicated, and incubated with viral particles that were premixed with 6 μg/ml DEAE-dextran at 25°C for 30 min (Sigma-Aldrich). After incubation at 37°C for 5 h with gentle mixing every hour, cells were centrifuged at 500 × g for 1 h and then 1000 × g for 4 min. Transduced B cells were cultured in virus-free FBS-RPMI in the presence of LPS plus IL-4 for 96 h and then harvested for flow cytometry analysis of expression of GFP (indicating expression of exogenous genes) and IgG1. Rosa26+/fl-STOP-fl-Ikkβca B cells expressed IKKβCA (as well as the GFP from the same bicistronic transcript) from the Rosa26 locus upon expression of the Cre recombinase and, consequently, deletion of the STOP cassette. Dead (7-AAD+) cells were excluded from analysis. To enforce expression of IKKβCA in plasma cells pregenerated from B cells stimulated with LPS plus IL-4, TGF-β, anti-δ/dex, and RA for 66 h, cells were transduced with pMIG or pMIG-Cre virus, as above, and harvested after 72 h to analyze by flow cytometry the expression of GFP (indicating transduced cells) and plasma cell proportion and viability.

Clinical and pathological analysis of MRL/Faslpr/lpr mice

Skin lesions, lymphadenopathy, and urine protein contents were assessed weekly. Skin lesions were scored on a scale of 0–4 as follows: 0, none; 1, mild (snout only); 2, moderate (<2 cm in snout, ears, and back); and 3, severe (>2 cm in snout and ears). Lymphadenopathy, as indicated by the appearances of lumps, was scored on a scale of 0–4 as follows: 0, none; 1, small (at one site); 2, moderate (at two sites); 3, large at three or more different sites; and 4, extremely large (at more than three sites). It was further confirmed by the increase in the size of brachial and inguinal lymph nodes when mice were sacrificed. Proteinuria was assessed and scored using semiquantitative Albustix strips (Bayer), which, despite giving a range of serum albumin levels for each scale (scale 0 for negative or trace, and 1–4), generated few false-positive results when the score was >3, as observed in virtually all 17-wk-old MRL/Faslpr/lpr mice. Splenomegaly was assessed when mice were sacrificed by the spleen size.

To assess kidney pathology, kidneys were fixed in Tissue-Tek OCT compound (Sankura Finetek) on dry ice. Seven-micrometer sections, as prepared by a cryostat (Leica), were fixed in cold acetone and stained with FITC-labeled anti-IgG1/anti-IgG2a mAbs. Sections were mounted using ProLong Gold with DAPI (Invitrogen) for confocal microscopic analysis. For periodic acid–Schiff (PAS) staining, kidneys were fixed in paraformaldehyde (3.6%) at room temperature for 72 h and, after washing twice with PBS, embedded in paraffin. Five-micrometer sections were prepared and processed with PAS stain. Glomerular change severity was graded based on glomerular activity, including glomerular cell proliferation (particularly mesangial matrix expansion), leukocyte infiltration, and cellular crescents. Mesangial matrix expansion was grades as follows: 0, no increase (matrix occupied up to 10% of the glomerulus); 1, mild (10–25%); 2, moderate (25–50%); and 3, marked (50–100%). The severity of interstitial mononuclear cell inltration was based on the ratio of infiltrated areas over the entire section (the value of area was determined by ImageJ software), quantified as the average of three sections 200 μm apart, and graded as follows: 1, mild; 2, moderate; 3, severe.

ELISA, ELISPOT, and anti-nuclear Ab assays

To determine titers of total IgM, IgG1, IgG2a, and IgG2b, sera were first diluted 4– to 128-fold with PBS (pH 7.4) plus 0.05% (v/v) Tween 20 (PBST). Two-fold serially diluted samples and standards for each Ig isotype were incubated in 96-well plates pretreated with sodium carbonate/bicarbonate buffer (pH 9.6) and coated with preadsorbed goat anti-IgM or anti-IgG (to capture IgG1, IgG2a, and IgG2b) Abs (all 1 mg/ml, Supplemental Table I). After washing with PBST, captured Igs were detected with biotinylated anti-IgM, anti-IgG1, anti-IgG2a, or anti-IgG2b Abs (Supplemental Table I), followed by reaction with HRP-labeled streptavidin (Sigma-Aldrich), development with o-phenylenediamine, and measurement of absorbance at 492 nm. Ig concentrations were determined using Prism (GraphPad Software) or Excel (Microsoft). To analyze titers of Ag-specific Abs (high-affinity NP binding or anti-dsDNA Abs), sera were diluted 1000-fold in PBST. Two-fold serially diluted samples were incubated in a 96-well plate preblocked with BSA and coated with NP4-BSA (in average four NP molecules on one BSA molecule) or dsDNA (10 μg/ml sonicated herring DNA). Captured Igs were detected with biotinylated Ab to IgM, IgG1, IgG2a, or IgG3. Data are relative values based on end-point dilution factors.

For ELISPOT analysis of total, NP-binding, and dsDNA-binding ASCs, MultiScreen filter plates (Millipore) were activated with 35% ethanol, washed with PBS, and coated with anti-IgM, anti-IgG, NP4-BSA, or dsDNA (all 5 μg/ml) in PBS. Single spleen or bone marrow cell suspensions were cultured at 50,000 cells/ml in FBS-RPMI supplemented with 50 μM 2-ME at 37°C for 16 h. After supernatants were removed, plates were incubated with biotinylated goat anti-mouse IgM, anti-IgG1, anti-IgG2a, or anti-IgG3 Ab, as indicated, for 2 h and, after washing, incubated with HRP-conjugated streptavidin. Plates were developed using the Vectastain AEC peroxidase substrate kit (Vector Laboratories). The stained area in each well was quantified using the CTL ImmunoSpot software (Cellular Technology) and depicted as the percentage of total area of each well for ASC quantification.

For semiquantitative anti-nuclear Ab (ANA) assays of sera from MRL/Faslpr/lpr and C57/Sle1Sle2Sle3 mice, sera were serially diluted in PBS (from 1:40 to 1:160), incubated on antinuclear Ab substrate slides (HEp-2 cell–coated slides, MBL Bion), and detected with a 1:1 mixture of FITC-labeled anti-IgG1 and anti-IgG2a mAbs (Supplemental Table I) following the manufacturer’s instructions. Images were acquired with an Olympus CKX41 fluorescence microscope.

Cytokine intracellular staining

Spleen cells were cultured in RPMI-FBS and stimulated with PMA (20 ng/ml) plus ionomycin (1 μg/ml; both from BioLegend) for 4 h at 37°C in the presence of brefeldin A. After pelleting, cells were stained with anti-CD4 mAb and fixable viability dye eFluor 450 followed by incubation with the BD Cytofix/Cytoperm buffer at 4°C for 20 min. After washing twice with the BD Perm/Wash buffer, cells were resuspended in HBSS with 1% BSA and stored overnight at 4°C. Cells were then stained with anti–IFN-γ and anti–IL-17 mAbs conjugated to different fluorophores in Perm/Wash buffer and analyzed by flow cytometry. Dead (eFluor 450+) cells were excluded.

Myeloid cell differentiation and function

Bone marrow cells (107) were cultured for 10 d in RPMI-FBS in the presence of conditioned media from L-929 cells that express M-CSF (which promotes differentiation into CD11b+ macrophages) or recombinant murine Flt3 ligand (which promotes differentiation into CD11c+ dendritic cells [DCs]; PeproTech), resulting in generation of >90% of CD11b+ or 20% of CD11c+ cells. After stimulation with TLR9 ligand CpG oligodeoxynucleotide for 1 h, cells were collected for RNA extraction and transcript analysis.

GST-RILP pulldown

RILP is an effector protein of activated Rab7 (Rab7-GTP). The E. coli strain BL21 harboring GST or GST-RILP–expressing vector, as previously described (23), was grown at 37°C to an OD of 0.6 before induced by isopropyl-1-thio-β-d-galactopyranoside (0.5 mM) at 30°C for 4 h to express GST or GST-RILP. After pelleting and washing with cold PBS, bacterial cells were resuspended in 5 ml of cold lysis buffer (25 mM Tris-HCl [pH 7.4], 1 M NaCl, 0.5 mM EDTA, 1 mM DTT, and 0.1% Triton X-100) supplemented with a protease inhibitor mixture (Sigma-Aldrich) and sonicated. Lysates were cleared and, after 5 ml of cold lysis buffer was added, incubated with 300 μl of pre-equilibrated slurry (50% packed volume) of glutathione–Sepharose 4B beads (GE Healthcare) at room temperature for 30 min. After washing with lysis buffer, beads (30 μl) with immobilized GST or GST-RILP were resuspended as slurry for protein quantification by the Bradford assay and pre-equilibrated in pull-down buffer (20 mM HEPES, 100 mM NaCl, 5 mM MgCl2, and 1% Triton X-100 plus protease inhibitor mixture). Beads were then incubated by rotating with lysates (300 μg) prepared from stimulated B cells in the pull-down buffer at 4°C overnight. After washing twice with cold pull-down buffer, bound proteins were eluted by incubation of beads in the SDS-PAGE sample buffer at 95°C for 10 min. Immobilized GST-RILP was analyzed by SDS-PAGE/immunoblotting using anti-GST mAb; Rab7-GTP was analyzed by immunoblotting using anti-Rab7 mAb.

Purification of recombinant Rab7 and nuclear magnetic resonance spectroscopy analysis

DNA sequence encoding Rab7 (human RAB7A amino acid residues 2–207) was synthesized using codon optimization for E. coli expression and cloned into the pET30a vector, which allows for expression of an N-terminal Strep-tag followed by a SUMO tag. After plasmid transformation, BL21(DE3) cells were grown at 37°C in minimum media until the culture OD600 reached 0.6. After the culture was transferred to 18°C, protein expression was induced by isopropyl-1-thio-β-d-galactopyranoside (1 mM) for 15 h. For protein purification, lysates were first subjected to Strep-Tactin affinity chromatography and then removal of the Strep-SUMO tag by digestion of SUMO protease at 4°C overnight, resulting in release of cleaved Rab7 (with an additional N-terminal Thr residue) into the supernatants. Trace amounts of cleaved tag were removed by a new round of Strep-Tactin affinity chromatography. Protein purity was >90%.

For nuclear magnetic resonance (NMR) spectroscopy analysis of free Rab7, Rab7 (0.3 mM) was exchanged into the buffer containing 20 mM NaPO4 (20 mM [pH 6.9]), NaCl (100 mM), and TCEP (1 mM) with 10% D2O. Two-dimensional 1H-15N heteronuclear single quantum coherence was acquired with 2048 and 128 complex points in the direct and indirect dimensions, respectively. For acquisition of the NMR spectrum of RAB7A in complex with CID 1067700, 2.5 μl of CID 1067700 stock solution (40 mM in nil) was added to 40 μl of RAB7A to yield final 1:8 protein/compound ratio, followed by NMR spectrum acquisition using the same parameters.

Docking analysis

All modeling studies were performed using the Schrodinger 2015-3 software suite and its graphical interface, Maestro. The crystal structure of the GTP-bound Rab7 (Protein Data Bank ID 1T91) was used as the docking target for CID 1067700. The bound GTP molecule and all water molecules were removed from the structure prior to docking. Docking was performed with Glide using first standard precision protocol to identify all possible poses with favorable Glide scores.

Chlamydia titration

For monitoring live organism shedding from swab samples, vaginal/cervical swabs were taken at the time points, as indicated. Each swab was soaked in 0.5 ml of sucrose-phosphate-glutamic acid and vortexed with glass beads to release chlamydial organisms into supernatants. IFUs were titrated on HeLa cell monolayers in duplicates. The infected cultures were processed for immunofluorescence assays. Briefly, inclusions were counted in five random fields per coverslip under a fluorescence microscope. For coverslips with <1 IFU per view field, IFUs on entire coverslips were counted. Coverslips showing obvious cytotoxicity of HeLa cells were excluded. The total number of IFUs was calculated based on the mean IFUs per view, the ratio of the view area to that of the well, the dilution factor, and inoculation volumes.

Flow cytometry, immunoblotting, cDNA synthesis and quantitative RT-PCR

These methods were as we previously described (7, 10, 22). Abs (Supplemental Table I) and oligonucleotides (Supplemental Table II) used had been validated to be specific. Flow cytometry data were analyzed using FlowJo software (Tree Star). For immunoblotting, signals were quantified by ImageJ (National Institutes of Health). For quantitative RT-PCR (qRT-PCR), the ΔΔCt method was used to determine levels of transcripts and data were normalized to levels of CD79B (human) or Cd79b (mouse), which encodes the BCR Igβ-chain that is constitutively expressed in B cells and, to a lesser degree, plasma cells.

Statistical analysis

Most statistical analyses were performed by a one-tailed type 1 (for pairwise comparison) or type 2 Student t test. Survival data were analyzed by the Mantel–Cox log-rank test. Chlamydia infection data were analyzed by the Wilcoxon rank sum test. A p value <0.05 was considered significant.

Results

Rab7 is highly expressed in human and mouse lupus B cells

Rab7 is upregulated in normal B cells by CSR-inducing stimuli (10), prompting us to analyze its expression in B cells from SLE patients and lupus mice. PBMCs from SLE patients expressed higher levels of RAB7A and AICDA transcripts than did healthy subjects (Fig. 1A). Likewise, B cells, including PNAhi B cells, from lupus-prone MRL/Faslpr/lpr and C57/Sle1Sle2Sle3 mice displayed elevated Rab7 and Aicda transcripts as compared with those from age-matched C57 counterparts (Fig. 1B, 1C). A similar difference was observed in Rab7 and AID protein levels in lymphoid organs (Fig. 1D). Thus, lupus B cells dysregulate Rab7 expression.

FIGURE 1.
FIGURE 1.

Rab7 is highly expressed in human and mouse lupus B cells. (A) qRT-PCR analysis of levels of RAB7A and AICDA transcripts in PBMCs isolated from lupus patients or healthy subjects (n = 9). Expression of RAB7B, which is unrelated to RAB7A and is not evolutionarily conserved, was comparable in these samples (data not shown). (B) qRT-PCR analysis of Rab7 and Aicda transcripts in spleen B cells isolated from 10-wk-old MRL/Faslpr/lpr mice and 36-wk-old C57/Sle1/Sle2/Sle3 mice. Data are expressed as ratios of values to those in B cells from age-matched C57 mice (mean and SD of data from three independent experiments). (C) qRT-PCR analysis of Rab7 and Aicda transcripts in sorted spleen CD19+PNAhi cells (>95% pure, which displayed higher Rab7 expression than CD19+PNAlo cells, not shown) from 10-wk-old MRL/Faslpr/lpr mice and NP-CGG–immunized C57 mice. Results are representative of two independent experiments (mean and SEM of triplicate samples). (D) Immunoblotting analysis of Rab7 and AID in spleen and lymph node cells isolated from 10-wk-old MRL/Faslpr/lpr and C57 mice as well as CH12 B cells.

Targeting of Rab7 by CID 1067700 inhibits NF-κB activation and AID induction in B cells

In view of treating lupus-prone mice with Rab7 inhibitor CID 1067700, we first tested the ability of this small molecule to inhibit CSR in vitro. CID 1067700 treatment of mouse B cells stimulated by CD154 or LPS plus IL-4 impaired Rab7 activation, as shown by reduced Rab7-GTP levels in RILP pull-down assays but normal Rab7 expression (Fig. 2A, 2B). This led to reduced AID expression and CSR to IgG1, despite normal B cell proliferation and germline IH-CH transcription, both of which are required for CSR (Fig. 2B–D). Consistent with the requirement of AID for CSR to all Ig isotypes, CID 1067700 inhibited induction of CSR to IgG, IgA, and IgE in human and mouse B cells (Fig. 2E–H).

FIGURE 2.
FIGURE 2.

CID 1067700 inhibits Rab7 activity, NF-κB activation, AID expression, and CSR in B cells. (A) GST-RILP pull-down analysis of Rab7-GTP (bottom panels) in B cells stimulated with LPS plus IL-4 for 48 h in the presence of nil or CID 1067700. Expression of total Rab7 was also analyzed (top panels). (B) Immunoblotting of Rab7, phosphorylated and total p65 in the canonical NF-κB pathway, p52 in the noncanonical NF-κB activation pathway, and AID and β-actin in stimulated B cells treated with nil or CID 1067700. Numbers on the right side indicate non-normalized values of quantified signals. (C) qRT-PCR analysis of levels of IgH germline Iμ-Cμ and Iγ1-Cγ1 transcripts, Aicda, postrecombination Iμ-Cγ1, and circle Iγ1-Cμ transcript in B cells stimulated with CD154 or LPS plus IL-4 and treated with different doses of CID 1067700. Data are expressed as ratios of values in CID 1067700–treated B cells to those in untreated cells (mean and SEM of data from triplicate samples). (D) Flow cytometry analysis of proliferation and CSR to IgG1 in CFSE-labeled B cells after stimulation in the presence of nil or CID 1067700 (top panels) and depiction of the proportion of switched IgG1+ cells in B cells that had completed each cell division (bottom panels). (E–H) CSR to different Ig isotypes in human or mouse B cells stimulated by different combinations of a T-dependent or T-independent primary CSR-inducing stimulus and a secondary inducing stimulus, as indicated, in the presence of nil or CID 1067700. (A), (B), and (E)–(H) are representative of three independent experiments.

CID 1067700 binds Rab7 with a high affinity (EC50: 10–20 nM), but it was also shown to bind several small GTPases, albeit with much lower affinity (i.e., with EC50 being at least 80 nM), and could affect functions of these GTPases in cell lines (16, 24). In our hands, it directly bound purified recombinant Rab7 (Fig. 3A), likely through two binding sites on the surface of Rab7, as suggested by computational docking of CID 1067700 onto the crystal structure of Rab7, with one site overlapping with the GTP binding site and the other located on the opposite face (Fig. 3B). In stimulated primary B cells, which upregulate Rab7 expression, CID 1067700 specifically targeted Rab7, as its inhibitory activity was abrogated when Rab7 was ablated in Igh+/Cγ1-creRab7fl/fl conditional knockout B cells (Fig. 3C), which per se showed reduced CSR (10). Conversely, CSR in CID 1067700–treated B cells was rescued by enforced expression of Rab7, but not Rab2a, a putative off-target of CID 1067700 (Fig. 3D). It was also increased by AID, which could rescue CSR in Rab7 knockout B cells (10).

FIGURE 3.
FIGURE 3.

CID 1067700 inhibits CSR in B cells by targeting Rab7. (A) NMR spectroscopy analysis of direct physical interaction of CID 1067700 with Rab7. Spectra of free Rab7 and Rab7/CID 1067700 are represented by red and green signals, respectively, with overlapping signals shown in black. Chemical shift perturbations and broadening observed for NMR signals (nonoverlapping signals) of multiple Rab7 residues are indicative of direct physical interaction. (B) Docking analysis of two candidate CID 1067700 binding sites on the opposite faces of Rab7 (left panel). The more favorable docking score (Glide score of −5.5) was obtained for CID 1067700 binding at the GTP-binding site of Rab7 (lower left panel) and the second binding site (Glide score of −2.7) located on the opposite face of Rab7 when CID 1067700 adopts a different conformation (lower right panel). The interaction interface within the GTP-binding site was slightly different from that predicted by others (16). (C) Flow cytometry analysis of CSR to IgG1 in B cells from Igh+/Cγ1-creRab7+/fl and Igh+/Cγ1-creRab7fl/fl littermates after stimulation with LPS plus IL-4 in the presence of nil or CID 1067700. Results are representative of three independent experiments. (D) CSR to IgG1 in B cells prestimulated with LPS in the presence of CID 1067700, transduced with pMIG or pMIG-Rab7, and then stimulated with LPS plus IL-4 for 72 h (left panels). The histogram (right panel) depicts CSR rescue by Rab7, Rab2a, a potential off-target of CID 1067700 with EC50 (170 nM) much higher than Rab7 (10–20 nM), or AID. Other potential off-targets (http://tinyurl.com/obgvd3v) are three small GTPases with EC50 comparable to Rab2a (i.e., H-Ras, 20–145 nM; Rac1, 80 nM; and Cdc42, 91–129 nM), much weaker targets (i.e., autophagy protein ATG4B, EC50 of 13 μM, and histone lysine methyltransferase G9a, EC50 of 39 μM), and growth factors/receptors (i.e., FGF22, GFER, VLA-4, and EGFR) that have not been independently verified as putative off-targets and, to the best of our knowledge, are not known to regulate CSR (mean and SEM of three independent experiments). (E) CSR to IgG1 in B cells isolated from Rosa26+/fl-STOP-fl-Ikkβca mice and transduced with pMIG or pMIG-Cre (which encoded the Cre recombinase to delete the STOP cassette in the Rosa26 locus and, therefore, allow for expression of IKKβCA) and then stimulated with LPS plus IL-4 in the presence of nil or CID 1067700 (mean and SEM of four experiments).

Similar to Rab7 deficiency, inhibition of Rab7 activity by CID 1067700 resulted in defective canonical NF-κB activation but normal noncanonical NF-κB activation (Fig. 2B). Expression of IKKβCA, a constitutively active mutant of IKKβ that can lead to canonical NF-κB hyperactivation (19), restored CSR in CID 1067700–treated B cells (Fig. 3E), indicating that the major defect in these cells is the impairment of canonical NF-κB activation. Thus, specific Rab7 inhibition in B cells hampers NF-κB activation, AID expression, and CSR induction without affecting B cell proliferation.

CID 1067700 blunts pathogenic autoantibody responses in lupus mice

The Rab7 upregulation in lupus B cells, together with the role of Rab7 in NF-κB activation, AID expression, and CSR, all of which are elevated in lupus B cells and implicated in lupus pathogenesis (25, 26), suggested that Rab7 inhibition would hamper NF-κB–dependent pathogenic autoantibody responses in vivo, for example, in MRL/Faslpr/lpr mice. These mice developed malar rash and dorsal skin lesions, lymphadenopathy, splenomegaly, and nephritis, leading to mortality as early as at 11 wk of age and an average life-span of ∼20 wk, with more than half of mice moribund between 17 and 23 wk of age (Fig. 4A, 4B). When treated with CID 1067700 for only 10 wk (starting at 10 wk, 1 wk before appearance of the first dead mouse in the untreated cohort), 14 of 16 MRL/Faslpr/lpr mice survived past the treatment period and half of mice were alive at 52 wk, compared with only one in the untreated cohort (Fig. 4A). Treated mice had normal body weight, rare skin lesions, and reduced lymphadenopathy and splenomegaly at 17 wk (Fig. 4B–D). They had some interstitial infiltration by monocytes (likely myeloid cells and T cells), but were largely free of glomerulonephritis, showing little proteinuria and decreased “crescent” formation and mesangial matrix expansion in glomeruli (Fig. 4E, 4F and data not shown).

FIGURE 4.
FIGURE 4.

Rab7 inhibition by CID 1067700 prevents disease development in lupus-prone mice. (A) Survival curve of 16 MRL/Faslpr/lpr mice treated with nil or CID 1067700. The survival of mice maintained in our facility was similar to that reported by The Jackson Laboratory and other groups. (B) Skin lesions in 26-wk-old nil- or CID 1067700–treated MRL/Faslpr/lpr mice. (C–F) Spleens and cervical lymph nodes (C), physiological metrics as indicated (D), indices of kidney damages (E), and PAS staining of kidney sections [(F), original magnification ×400] in 17-wk-old MRL/Faslpr/lpr mice treated with nil or CID 1067700 [(C) and (F), representative of three independent experiments; (D) and (E), mean and SD of four pairs].

Lupus nephritis is tightly associated with and/or caused by deposition of immunocomplexes that contain class-switched IgG autoantibodies in the kidney. IgG-immunocomplex deposition was reduced in MRL/Faslpr/lpr mice treated with CID 1067700 (Fig. 5A). Serum levels of IgG ANAs were also decreased in treated MRL/Faslpr/lpr and C57/Sle1Sle2Sle3 mice (Fig. 5B). As shown by time-course analyses of autoantibodies that bound dsDNA (anti-dsDNA), the ratio of pathogenic IgG classes, for example, IgG1, IgG2a, and IgG2b, over the nonpathogenic IgM class (IgG/IgM, depicted as RIgG) remained low in treated mice despite variations of IgG and IgM titers in different mice in the same cohort (Fig. 5C and data not shown). So did RIgG for total IgG, in contrast to the steadily increased anti-dsDNA and total RIgG, until peaking, in untreated mice (Fig. 5C). Thus, inhibition of Rab7 suppresses IgG autoantibody responses and prevents development of disease symptoms in lupus mice, leading to significantly increased life-span.

FIGURE 5.
FIGURE 5.

Rab7 inhibition blocks generation of pathogenic autoantibodies. (A) IgG-immunocomplex formation in the kidney of 17-wk-old nil- or CID 1067700–treated MRL/Faslpr/lpr mice (representative of kidney sections in three independent experiments). (B) ANA levels in serum samples (diluted 40-fold) from MRL/Faslpr/lpr mice, as in (A), and 54-wk-old nil- or CID 1067700–treated C57/Sle1Sle2Sle3 mice. ANAs to different autoantigens, as shown by different patterns of fluorescence images at higher magnifications, were all reduced in CID 1067700–treated mice (representative of three independent experiments). More diluted serum samples, for example, by 160-fold, from CID 1067700–treated mice showed virtually no signals (data not shown). (C) Ratios of anti-dsDNA (top panels) and total (bottom panels) IgG1, IgG2a, and IgG2b titers to titers of the IgM counterparts in nil- or CID 1067700–treated MRL/Faslpr/lpr mice at different ages, as indicated. Data were normalized to values in mice at the age of from 10 wk (set as 1), right before the treatment started, and were depicted as RIgG1, RIgG2a, and RIgG2b (mean and SEM of four mice per group). IgG3 titers were generally low (data not shown).

CID 1067700 targets B cells and specifically impairs the CSR machinery in vivo

The reduction of RIgG suggested that the CSR process was inhibited in CID 1067700–treated MRL/Faslpr/lpr mice. Indeed, these mice showed unchanged numbers of B cells but significantly decreased IgG+ B cells, concomitant with a slightly increased proportion of unswitched IgM+ B cells (Fig. 6A). Decreased CSR, as also shown by reduced postrecombination Iμ-Cγ transcripts (the molecular indicators of completed CSR), was associated with lower expression of CSR machinery genes, such as Aicda and 14-3-3γ, which is important for the targeting of AID to IgH switch (S) regions and is induced in an NF-κB–dependent manner (22, 27, 28); Rab7 expression was also slightly reduced (Fig. 6B). Expression of the A20 (Tnfaip3) gene, which dampens NF-κB activation and controls autoimmunity (29), or germline Iμ-Cμ transcription, however, was not affected, nor was expression of Prdm1 (encoding Blimp-1), Xbp1, which is essential for the plasma cell secretion function, or genes (Vps34, LC3, and Becn1) implicated in autophagy, which regulates plasma cell differentiation/functions (30, 31). Treated B cells were normal in proliferation, CD21/CD35 expression, induction of the CD80 activation markers, and expression of CD40 and MHC class II, which are important for B cells to interact with CD4+ Th cells (Fig. 6C), thereby emphasizing the inherent nature of the CSR machinery defect.

FIGURE 6.
FIGURE 6.

Rab7 inhibition hampers CSR in lupus-prone mice. (A) Flow cytometry analysis of number of B cells (left panel) and proportion of IgM+, IgG1+, and IgG2a+ cells among B cells (right panels) in 17-wk-old nil- or CID 1067700–treated MRL/Faslpr/lpr mice (mean and SD of four pairs of mice). The proportion of these B cells was increased due to the reduced splenomegaly (data not shown). Both nil- and CID 1067700–treated MRL/Faslpr/lpr mice had B lymphocytopenia, with an average of 3.2 × 107 CD19+ B cells per spleen, as compared with ∼5.5 × 107 B cells in age-matched C57 mice (data not shown). Moribund MRL/Faslpr/lpr mice were excluded from analyses due to their severe B lymphocytopenia. (B) Expression of different transcripts, as indicated, in CD19+ B cells sorted from MRL/Faslpr/lpr mice, as in (A). Data were depicted as the ratio of values in CID 1067700–treated mice to those in nil-treated mice (*p < 0.05, **p < 0.01). (C) Flow cytometry analysis of B cell expression of markers (left and middle panel sets), as indicated, and proliferation (right panel set) in MRL/Faslpr/lpr mice, as in (A).

Rab7 has been suggested to be expressed in myeloid cells, in addition to B cells (32), prompting us to analyze the impact of Rab7 inhibition on CD11b+ macrophages and CD11c+ DCs from MRL/Faslpr/lpr mice. The proportions of these cells, their survival in vitro, and induced expression of cytokines that could influence B cells and autoantibody responses, such as BAFF and type I IFN (IFN-α and IFN-β), were not affected by CID 1067700 treatment, with the only exception of reduced Il1b expression in CD11c+ DCs (Fig. 7A–C). Reflecting the low expression of Rab7 in T cells and a modest role of this molecule in T cell functions (33), CID 067700 treatment did not change the proportions of total CD4+ T cells and those producing IFN-γ, which is critical for CSR to IgG2a and important in systemic autoimmunity (34–36), in MRL/Faslpr/lpr mice or survival of CD4+ lupus T cells in vitro (Fig. 7C, 7D). This, together with the ability of CID 1067700–treated C57 mice to clear Chlamydia infection, which is dependent on T cells, but not on B cells or plasma cells (37, 38), showed that this small molecule does not have a major impact on T cells (Fig. 7E). Thus, CID 1067700 selectively targets B cells and impairs the CSR machinery in lupus-prone mice without altering proliferation/activation of B cells.

FIGURE 7.
FIGURE 7.

Rab7 inhibition does not affect myeloid cells or T cells. (A) Flow cytometry analysis of CD11b+ and CD11c+ cells in 17-wk-old nil- or CID 1067700–treated MRL/Faslpr/lpr mice. (B) Expression of cytokine-encoding genes, as indicated, in CD11b+ and CD11c+ cells generated from the bone marrow cells isolated from untreated 10-wk-old MRL/Faslpr/lpr mice and then stimulated by CpG oligodeoxynucleotide in vitro in the presence of nil or CID 1067700 (mean and SD of triplicate samples, *p < 0.05). (C) Survival of CD11b+ and CD11c+ myeloid cells as well as CD4+ T cells isolated from untreated 10-wk-old MRL/Faslpr/lpr mice and cultured in vitro in the presence of nil or different doses of CID 1067700 for 24 or 48 h (mean and SD of triplicate samples). (D) Flow cytometry analysis of proportion of CD4+ T cells and expression of IFN-γ in CD4+ T cells in nil- or CID 1067700–treated MRL/Faslpr/lpr mice, as in (A). Expression of IL-17 is low despite the important role of this cytokine in lupus (70). Consistent with reduced splenomegaly, the number of T cells was reduced, possibly as the secondary effect of reduced activity of autoreactive B cells. No CD19+ cells expressed IFN-γ or IL-17 (data not shown). (E) Shedding of Chlamydia in C57 mice treated with nil or CID 1067700 (mean and SEM of five mice in each group). B cells are not required for the clearance of primary Chlamydia infection. (A), (C), and (D) are representative of three independent experiments.

Rab7 deficiency or Rab7 inhibition reduces ASCs

Consistent with the surge of IgG+ ASCs during lupus flare, most cells expressing the plasma cell marker CD138 in MRL/Faslpr/lpr mice with active lupus (e.g., at 17 wk) were IgG+, as shown by intracellular staining (Fig. 8A). Upon Rab7 inhibition, IgG+ plasma cells were greatly decreased (due to defective CSR) with a concomitant increase in the proportion of IgM+ plasma cells. However, the number of ASCs that produced IgM autoantibodies (e.g., anti-dsDNA) remained unchanged (Fig. 8B). This together with the lower number of IgG1+ and IgG2a+ ASCs indicated an overall reduction in ASCs upon treatment with CID 1067700. This Rab7 inhibitor had a similar effect on ASCs in C57 mice, as indicated by the reduced Ag-specific IgG1+ ASCs and unchanged IgM+ ASCs in the spleen of mice injected with a T-dependent (NP-CGG) or T-independent (NP-LPS) Ag, which resulted in virtual abrogation of circulating Ag-specific IgGs (Fig. 8C). Likewise, Rab7 knockout in B cells and their plasma cell progenies, as occurring in Igh+/Cγ1-creRab7fl/fl mice, reduced total and Ag-specific IgG1+ ASCs [our previous findings (10)] with no increase in IgM+ ASCs (Fig. 8D). ASCs in the bone marrow, in which long-lived plasma cells take residence (39), were even more sensitive to Rab7 inhibition or knockout, as both IgG+ and IgM+ ASCs were decreased (Fig. 8C, 8D). Finally, IgG+ ASCs preformed in vivo were vulnerable to CID 1067700 treatment, as they failed to produce Abs ex vivo (Fig. 8E). Thus, abrogation of Rab7 expression or inhibition of its activity in normal and lupus-prone mice affects ASC production/maintenance, thereby compounding the defect in CSR to suppress class-switched Ab and autoantibody responses.

FIGURE 8.
FIGURE 8.

Rab7 deficiency or inhibition impairs ASC production/functions in vivo and in vitro. (A) Levels of intracellular IgM, IgG1, and IgG2a in spleen CD138+ cells from 17–wk-old nil- or CID 1067700–treated MRL/Faslpr/lpr mice (representative of three independent experiments). (B) ELISPOT analysis of IgM+, IgG1+, and IgG2a+ ASCs in 17-wk-old nil- or CID 1067700–treated MRL/Faslpr/lpr mice (right panels, mean and SEM of four pairs of mice). (C) ELISPOT analysis of IgM+ and IgG+ (IgG1 or IgG3, as indicated) NP-binding ASCs in the spleen and bone marrow, as well as titers of circulating NP-binding IgM and IgG1 or IgG3 Abs, in nil- or CID 1067700–treated C57 mice immunized with NP-CGG (top panels) or NP-LPS (bottom panels) (mean and SEM of three pairs of mice). (D) ELISPOT analysis of IgM+ NP binding and total ASCs in the spleen and bone marrow in Igh+/Cγ1-creRab7fl/fl mice and their Igh+/Cγ1-creRab7+/fl littermates immunized with NP-CGG (mean and SEM of three pairs of mice). (E) ELISPOT analysis of IgG1+ ASCs isolated form the bone marrow of C57 mice and treated in vitro with nil or CID 1067700 for 24 h before incubation for 24 h in the presence of nil or CID 1067700, totaling 48 h treatment (mean and SEM of three independent experiments).

Rab7 inhibition impairs plasma cell survival

Despite their impact on ASCs (consisting of plasmablasts and plasma cells), neither CID 1067700 nor Rab7 knockout affected in vitro generation of CD138hi cells upon induction of B cells by all tested stimuli, such as LPS plus IL-4, TGF-β, BCR crosslinking, and RA, which led to >70% of live cells being CD138hi (Fig. 9A and data not shown). This suggested that Rab7 was not involved in plasma cell generation and prompted us to analyze the effect of CID 1067700 on plasma cell survival. In MRL/Faslpr/lpr mice, CID 1067700 treatment resulted in increased death and reduced numbers of CD138hi cells (Fig. 9B, 9C). Even residual live CD138hi cells expressed lower levels of Cxcr4, Il6r, and Vla4, all of which encode surface receptors that mediate plasma cell survival; in contrast, expression of Bcma and Taci, which are important for APRIL-dependent survival, was not affected (Fig. 9D). Expression of intracellular factors important for plasma cell survival (such as Irf4, Mcl1, and Hdac11) as well as the Atg5 autophagy gene was also affected. Expression of Prdm1 or Xbp1, which are important to maintain the plasma cell secretory function but dispensable for their survival (40), however, was not affected. To further confirm that Rab7 inhibition directly affects plasma cell survival, we treated CD138hi cells generated in vitro with CID 1067700. These cells displayed high levels of early and late apoptosis, as compared with the much slower loss of viability in their untreated counterparts (Fig. 9E). Enforced NF-κB activation through IKKβCA expression prevented CD138hi cells from being killed by CID 1067700 (Fig. 9F), showing that Rab7 plays a role in the plasma cell survival in vivo and in vitro, in part by mediating NF-κB activation.

FIGURE 9.
FIGURE 9.

Rab7 deficiency or inhibition impairs the survival of plasma cells, but not their generation. (A) Generation of CD19−/loCD138hi plasma cells in vitro from Igh+/Cγ1-creRab7fl/fl B cells and their Igh+/Cγ1-creRab7+/fl B cell counterparts upon stimulation by LPS plus IL-4 or IL-4, TGF-β, anti-δ/dex, and RA (left panels) as well as from C57 B cells undergoing same stimulation and treated with nil or CID 1067700 (right panels). (B) Flow cytometry analysis of total (TCR−CD19−/lo)CD138hi cells in 17-wk-old nil- or CID107700–treated MRL/Faslpr/lpr mice (top panels) and proportion of dead (7-AAD+) cells (bottom panels). The proportion of (TCR−CD19+CD138−) B cells was increased, but their number and viability were not changed (data not shown). Results are representative of three independent experiments. (C) Flow cytometry analysis of the number of live (TCR−CD19−/lo)CD138hi cells in MRL/Faslpr/lpr mice, as in (B) (mean and SD of four pairs of mice). (D) Expression of different transcripts, as indicated, in live 7-AAD−CD138hi cells sorted from 17-wk-old nil- or CID 1067700–treated MRL/Faslpr/lpr mice. Similar to Cd35 and Cd44 (which do not promote plasma cell survival), Vla4 and Cxcr4 are also involved in plasma cell homing to the bone marrow. Expression of iNos, as suggested to promote plasma cell survival in normal mice, was not detectable. Decreased expression of Iμ-Cγ1 and Iμ-Cγ2a in plasma cells was expected owing to reduced CSR. Data were depicted as the ratio of values in CID 1067700–treated mice to those in nil-treated mice (mean and SD of four pairs of mice, *p < 0.05). (E) Flow cytometry analysis of early (annexin V+7-AAD−) and late (annexin Vhi7-AAD+) apoptosis in pregenerated CD19–/loCD138hi plasma cells (after stimulation by LPS plus IL-4, IL-5, TGF-β, anti-δ/dex, and RA for 66 h) after treatment with nil or CID 1067700 for 24, 48, and 72 h (the histogram depicts the mean and SEM of three independent experiments; death of residual CD19+ B cells in the same culture was not affected by CID 1067700). Flow cytometry data (top panels) are representative of three independent experiments. (F) Survival of CD19−/loCD138hi plasma cells pregenerated in the presence of pMIG-Cre retrovirus to express IKKβCA and then treated with nil or CID 1067700 for 72 h. Results are representative of three independent experiments.

Discussion

Stemming from our previous findings (10), data reported in this study have further outlined an important role of Rab7 in Ab and autoantibody responses, owing to its functions in modulating Aicda expression, B cell class switching, and plasma cell survival. Despite the partial impairments of these processes by Rab7 inhibition or knockout, their combined outcome was the virtual abrogation of class-switched specific Abs in normal mice and paucity of pathogenic autoantibodies in lupus-prone mice. As we showed, small molecule Rab7 inhibition hampered CSR induced by all primary stimuli, including the one (CD154) produced by T cells, which mediate many autoimmune conditions in part by dysregulating B cells, and a ligand (R-848) of TLR7, an endosomal TLR that strongly promotes lupus pathogenesis and does so in a B cell–intrinsic manner (41). Thus, Rab7, which is an endosome-tethered protein, participates in B lineage cell differentiation processes initiated by many immune receptors irrespective of their initial locations, for example, CD40 and TLR4 on the surface and TLR7 in the endosome. Surface immune receptors, however, need to be internalized for Rab7 to mediate CSR and plasma cell survival, likely in a manner dependent on Rab7+ endosomes.

As indicated by our results showing IKKβCA-mediated rescue of CSR and plasma cell survival in CID 1067700–treated cells, the B cell– and plasma cell–intrinsic roles of Rab7 are, at least in part, through activation of NF-κB, a transcription factor central to B lineage cell functions (42, 43). Rab7-dependent NF-κB activation, however, is dispensable for B cell development and differentiation into plasma cells, as suggested by unchanged numbers of CD19+ B cells in CID 1067700–treated mice in vivo and generation of plasma cells from CID 1067700-treated B cells or Rab7 knockout B cells in vitro. It would also be absent during early activation of naive B cells upon CD40 or TLR engagement, as these cells express low levels of Rab7 and contain sparse intracellular membrane structures. Only after Rab7 upregulation, likely together with expansion of the intracellular membrane network, would Rab7 nucleate the assembly of putative “intracellular signalosomes” on different membrane types (e.g., endosomes in B cells and autophagosomes in plasma cells)—through mechanisms that are not yet clear—for sustained NF-κB activation. This would be required for efficient induction of AID, whose expression peaks at 48 h after B cell stimulation, and for continuous expression of genes in plasma cells that maintain cell survival. In contrast, Rab7-independent early NF-κB activation would occur through different signalosomes, for example, those on the plasma membrane in B cells with cell surface receptor engagement. It also plays a role in the Rab7 gene transcription, possibly through several κB sites in the promoter region (H. Zan, P. Casali, and Z. Xu, unpublished observations).

Rab7 is inherently the preferred target of CID 1067700, as shown by its much lower EC50 as compared with the few small GTPase off-targets of this compound (16). As suggested by our docking analyses, CID 1067700 could potentially bind at two distinct sites on the Rab7 surface. Existence of a candidate binding site that is different from the GTP-binding site (the site that also exists in potential off-targets of CID 1067700) would, at least in part, explain the Rab7-specific inhibitory activity of CID 1067700. This together with upregulated Rab7 expression in B cells (and likely in plasma cells), including in PNAhi germinal center B cells in immunized mice, would underpin the specific inhibition of Rab7 by CID 1067700, as more molecules would be available for targeting. Likewise, the dysregulated Rab7 expression would make lupus B cells preferred targets of CID 1067700. Such dysregulation may be the consequence of a putative positive-feedback loop involving NF-κB hyperactivation (25, 26). It could also be exacerbated by female hormones, as suggested by enhancement of Rab7 expression by estrogens (H. Zan, et al., unpublished observations). This together with estrogen upregulation of AID expression through the HoxC4 homeodomain transcription factor would contribute to the female bias of autoantibody-mediated systemic autoimmunity (11, 44, 45). In the subset of human lupus B cells that displayed moderate Rab7 expression, as shown in our studies, NF-κB hyperactivation may be through alternative mechanisms, such as A20 downregulation (29).

Rab7 inhibition reduced expression of several genes instrumental to plasma cell survival, such as Cxcr4, Irf4, and Mcl1 (40, 46, 47). Expression of CXCR4 (a G protein–coupled receptor) has been shown to be boosted by NF-κB, consistent with the identification of several κB sites in the Cxcr4 gene locus (48), and a role of Rab7-dependent NF-κB activation in plasma cell survival. The presence of several Irf4-binding sites in the Cxcr4 promoter (49) also suggests that Cxcr4 transcription is regulated by Irf4, whose expression would be mediated by Rab7 (shown here) and promoted by NF-κB (50). Expression of Mcl1 in plasma cells is in general lower than that in B cells (51), perhaps explaining higher sensitivity of plasma cells to death upon Rab7 inhibition. Rab7 deficiency or inhibition in mice may also impair the autophagy-dependent Ab secretion, which together with decreased plasma cell survival would lead to marked reduction in ASCs. Similar to anti-dsDNA–specific ASCs in the spleen, kidney resident plasma cells that secrete pathogenic nephrophilic Abs (52–54) may be reduced, leading to significantly reduced kidney damage.

As a small molecule, CID 1067700 injected i.p. was “metabolized” in vivo and required weekly administration to maintain detectable levels in the circulation and lymphoid organs (D.V. Kulp, C.R. Rivera, P. Casali, and Z. Xu, unpublished observations). Despite this, Rab7 inhibition by CID 1067700 could prevent development of lupus pathology in MRL/Faslpr/lpr mice and extend the lifespan by >32 wk, after a treatment period of only 10 wk, suggesting that long-lasting changes had occurred in treated B cells—treated plasma cells would die. These changes likely include alterations in the epigenome in lupus B cells with extended survival (due to the Fas/lpr mutation) as well as changes in the BCR repertoire and/or the memory autoreactive B cell compartment. Putative epigenetic changes might occur through downregulation of NF-κB–dependent expression of histone-modifying enzymes and/or rebalance of exaggerated Rab7-dependent autophagy, which modulates the microRNA machinery (15, 55). B cell–intrinsic mechanisms may be further complemented by changes in regulatory immune elements, such as T regulatory cells and DCs (56), possibly including altered type I IFN gene signatures in plasmacytoid DCs, as short-term plasmacytoid DC ablation could also elicit long-term prevention of nephritis in the BXSB lupus mouse mode (57). Such DC changes, however, would be indirect, as CID 1067700 did not decrease cytokine production by DCs in vitro. High Rab7 expression levels in DCs, however, do suggest that Rab7 regulates certain DC functions, perhaps Ag/autoantigen presentation through autophagy induction (58) and MyD88 signaling, which mediates skin lesions (59). In contrast, Rab7 is largely dispensable for T cell survival and functions (e.g., in clearing primary Chlamydia infection, as shown in the present study). Addressing specific roles of Rab7 in these and other immune compartments requires generation of different conditional knockout mice.

In contrast to the nondiscriminating B cell–depletion lupus therapeutics approaches, CID 1067700 or its future derivatives with improved pharmacodynamics and pharmacokinetics properties would maintain IgM+ B cells and protective IgMs. It is conceivable that a Rab7 inhibitor can be combined with anti-BAFF or anti-CD20 mAb toward better therapeutic effects (60–62). Such an inhibitor may also be combined with a histone deacetylase inhibitor to abrogate plasma cells by affecting plasma cell survival and generation, respectively: synthetic and naturally occurring (e.g., butyrate, a metabolite of gut microbiota) histone deacetylase inhibitors blunt Ab and autoantibody responses by inhibiting CSR/SHM and B cell differentiation into plasma cells, but not plasma cell survival (3, 21). The translational implications of Rab7-dependent NF-κB activation extend beyond lupus. Rab7 may promote B cell lymphomagenesis mediated by NF-κB hyperactivation (63–65), as suggested by the frequent DNA insertions/deletions and chromosomal translocations in/around the human RAB7A locus on the chromosome 3 (3q21) in hematologic malignancies (66–68). It may also play a role in NF-κB–mediated survival of multiple myelomas (69), as it does in maintaining the survival of plasma cells. Addressing these possibilities would greatly expand our studies toward the understanding of the role of Rab7 in immune regulation.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Crystal Lafleur for technical help, Tian Shen for help in plasma cell induction, Egest J. Pone and Christie-Lynn Mortales for ELISPOT, Tamara McRae for PAS kidney histology analysis, Dr. Dmytro Kovalskyy for docking analyses, Dr. Benjamin J. Daniel and Karla M. Gorena in the University of Texas Health Science Center at San Antonio Flow Core Facility for help with cell sorting, and the University of Texas Health Science Center at San Antonio NMR and Analytical Ultracentrifugation Core Facility (supported in part by National Institutes of Health Grant P30 CA054174 to the Cancer Therapy and Research Center of University of Texas Health Science Center at San Antonio) for NMR analysis. We thank Dr. Aimee L. Edinger for the RILP-expressing construct, Dr. Nu Zhang for help with cytokine intracellular staining, and Dr. Xiaodong Li for help with myeloid cell differentiation assays.

Footnotes

  • ↵3 P.C. and Z.X. jointly directed the study.

  • This work was supported by National Institutes of Health Grants AI 079705 and AI 105813 (to P.C.), AI 124172 (to Z.X.), AI 104476 (to D.N.I.), and AI 047997 (to G.Z.). P.C. was also partially supported by Alliance for Lupus Research Target Identification in Lupus Grant ALR 295955 and the Zachry Foundation Distinguished Chair, D.N.I. by a Voelcker Fund Young Investigator Award, H.Z. by an Arthritis National Research Foundation research grant, and R.W. by the Xiangya School of Medicine, Central South University of China.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    7-AAD
    7-aminoactinomycin D
    AID
    activation-induced cytidine deaminase
    ANA
    anti-nuclear Ab
    ASC
    Ab-secreting cell
    C57
    C57BL/6
    CGG
    chicken γ-globulin
    CSR
    class switch DNA recombination
    DC
    dendritic cell
    NMR
    nuclear magnetic resonance
    NP
    (4-hydroxy-3-nitrophenyl)acetyl
    PAS
    periodic acid–Schiff
    PNA
    peanut agglutinin
    qRT-PCR
    quantitative RT-PCR
    RA
    retinoic acid
    Rab7
    Ras-related in brain 7
    SLE
    systemic lupus erythematosus.

  • Received August 16, 2016.
  • Accepted September 9, 2016.

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