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. 2021 Aug;35(8):e21785.
doi: 10.1096/fj.202100047R.

Cf-02, a novel benzamide-linked small molecule, blunts NF-κB activation and NLRP3 inflammasome assembly and improves acute onset of accelerated and severe lupus nephritis in mice

Shin-Ruen Yang  1   2 , Kuo-Feng Hua  3   4 , Chih-Yu Yang  5   6 , Ann Chen  2 , Jui-Chun Weng  7 , Yu-Ling Tsai  2 , Chih-Jun Wan  1 , Chung-Yao Wu  2 , Chia-Chung Lee  8 , Jia-Feng Chan  9 , Chih-Yu Hsieh  9   10 , Yu-Juei Hsu  11 , Chia-Chao Wu  11 , Debabrata Mukhopadhyay  12 , Hsu-Shan Huang  8   13 , Feng-Cheng Liu  14 , Shuk-Man Ka  7
Affiliations

Cf-02, a novel benzamide-linked small molecule, blunts NF-κB activation and NLRP3 inflammasome assembly and improves acute onset of accelerated and severe lupus nephritis in mice

Shin-Ruen Yang et al. FASEB J. 2021 Aug.

Abstract

In the present study, acute onset of severe lupus nephritis was successfully treated in mice using a new, benzamide-linked, small molecule that targets immune modulation and the NLRP3 inflammasome. Specifically, 6-(2,4-difluorophenyl)-3-(3-(trifluoromethyl)phenyl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione (Cf-02) (a) reduced serum levels of IgG anti-dsDNA, IL-1β, IL-6, and TNF-α, (b) inhibited activation of dendritic cells and differentially regulated T cell functions, and (c) suppressed the NF-κB/NLRP3 inflammasome axis, targeting priming and activating signals of the inflammasome. Moreover, treatment with Cf-02 significantly inhibited secretion of IL-1β in lipopolysaccharide-stimulated macrophages, but this effect was abolished by autophagy induction. These results recommend Cf-02 as a promising drug candidate for the serious renal conditions associated with systemic lupus erythematosus. Future investigations should examine whether Cf-02 may also be therapeutic in other types of chronic kidney disease involving NLRP3 inflammasome-driven signaling.

Keywords: NF-κB/NLRP3 inflammasome axis; autophagy; benzamide-linked small molecule; severe lupus nephritis.

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Conflict of interest statement

CONFLICT OF INTEREST

Each author of this work declares no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
LPS-binding protein and IL-1β levels in SLE patients. A, Serum levels of LPS-binding protein. B, IL-1β secretion from PBMCs treated with LPS and ATP, followed by treatment with Cf-02. Data are presented as means ± SEM. Cf-02, a novel benzamide-linked small molecule; HC, healthy control; PBMCs, peripheral blood mononuclear cells; SLE, systemic lupus erythematosus; SS, Sjögren syndrome. **P < .01, ***P < .005, ****P < .001
FIGURE 2
FIGURE 2
Renal function, albuminuria, pathology, neutrophil influx, IgG anti-dsDNA, and cytokines in ASLN mice. Serum levels of (A) BUN and (B) Cr; (C) Urine albumin levels; (D) Renal pathology, H&E staining. The arrows indicate mononuclear leukocyte infiltration. The arrowheads indicate neutrophil infiltration. E-I, Scoring for renal pathology; (J) Scoring for glomerulonephritis activity. K, Collagen IV by IHC staining. L, Scoring for IHC. M, Neutrophil influx was quantified in MSU crystals-induced peritonitis in mice by flow cytometry. N, Serum levels of IgG anti-dsDNA autoantibody; Serum levels of (O) IL-1β, (P) IL-6 and (Q) TNF-α. Scale bars = 50 μm. Original magnification 400×. Data are presented as means ± SEM for seven mice per group. ASLN, accelerated, severe lupus nephritis; BUN, blood urea nitrogen; Cf-02, a novel benzamide-linked small molecule; Cr, creatinine. ***P < .005, ****P < .001, #Not detectable. ns, no significant difference
FIGURE 3
FIGURE 3
Immunohistochemistry, real-time PCR assay, flow cytometry in ASLN mice and OT-II antigen-specific T cell proliferation analysis. A, F4/80+ macrophages infiltrating the kidney; (B) CD3+ T cells infiltrating the kidney; (C) CD11c+ dendritic cells infiltrating the kidney. IHC, Original magnification, 400×. The arrows indicate mononuclear leukocyte infiltration; (D-F) Scoring for IHC. Renal mRNA levels of (G) M1 and (H) M2 by real-time PCR assay. I, CD4+CD44hiCD62Llo T memory cells; (J) CD8+CD44hiCD62Llo-hi T memory cells; (K) CD4+CD25+Foxp3+ Treg cells. Flow cytometry using splenocytes. L, T cell proliferation analysis using splenocytes by [H3]-thymidine incorporation assay. Data are presented as means ± SEM with seven mice per group. M, CD11c+CD80+ and (N) CD11c+CD86+ in BMDCs determined by flow cytometry. O, OT-II antigen-specific T cell proliferation analysis using [H3]-thymidine incorporation assay. A 1:4 ratio of BMDCs to CD4+ T cells was used. ASLN, accelerated, severe lupus nephritis; BMDCs, bone marrow derived dendritic cells; Cf-02, a novel benzamide-linked small molecule; MFI, mean fluorescent intensity. *P < .05, **P < .01, ***P < .005 and ****P < .001. #Not detectable. ns, no significant difference
FIGURE 4
FIGURE 4
Renal ROS production and NLRP3 inflammasome activation in ASLN mice. A, ROS. B, NF-κB activity. C, mRNA levels of NLRP3, caspase-1, IL-1β. D, Protein levels NLRP3, caspase-1 and IL-1β; (E-H) Semiquantitative analysis. I, Caspase-1 activity. Data are presented as means ± SEM with seven mice per group. ASLN, accelerated, severe lupus nephritis; Cf-02, a novel benzamide-linked small molecule; ROS, reactive oxygen species. **P < .01, ***P < .005 and ****P < .001. #Not detectable. ns, no significant difference
FIGURE 5
FIGURE 5
NLRP3 inflammasome activation in macrophages. A, IL-1β by ELISA. B, IL-1β, IL-18, (C) Caspase-1, (D) NLRP3 and ASC by Western blot analysis. Cells were incubated with 1 μg/mL of LPS for 5.5 hours, and then with Cf-02 at concentrations indicated for 30 minutes, followed by 5 mM ATP for 30 minutes. Data are presented as means ± SEM for three separate experiments, and each experiment was performed in triplicate. Casp-1, caspase-1; Cf-02, a novel benzamide-linked small molecule. ***P < .005 and ****P < .001. ns, no significant difference
FIGURE 6
FIGURE 6
Phosphorylation of ERK1/2, JNK1/2, p38MAPK, I-κB, NLRP3 assembly and ASC oligomerization in macrophages. A, Protein levels of NLRP3, proIL-1β; (B) p-ERK1/2, p-JNK1/2, p-p38MAPK and (C) p-I-κB by Western blot analysis. Cells were incubated with Cf-02 at indicated concentrations for 30 minutes, followed by incubation with 1 μg/mL of LPS. NLRP3 assembly of (D) ASC-NLRP3; (E) NEK7-NLRP3; (F) PKR-NLRP3 by immunoprecipitation. G, ASC oligomerization by Western blot analysis. Cells were incubated with 1 μg/mL of LPS for 5.5 hours, and then with Cf-02 at concentrations indicated for 30 minutes, followed by 5 mM ATP for 30 minutes. Data are presented as means ± SEM with three separate experiments, and each experiment was performed in triplicate. Cf-02, a novel benzamide-linked small molecule; IB, immunoblotting; IP, immunoprecipitation
FIGURE 7
FIGURE 7
Activated autophagy-mediated inhibition of the NLRP3 inflammasome in macrophages. A, Protein levels of LC3B-I/II and p62 at a resting stage by Western blot analysis. Cells were incubated with Cf-02 at concentrations indicated in a time-dependent manner. B, Protein levels of LC3B-I/II and p62 by Western blot analysis. Cells were incubated with Cf-02 for 6 hours, and with or without 3-MA for 30 minutes (5 mM; an autophagy inhibitor). Protein levels of IL-1β by (C) ELISA and (D) Western blot analysis. Cells were incubated with 1 μg/mL of LPS for 5.5 hours, with or without 3-MA (5 mM) for 30 minutes, and Cf-02 for 30 minutes, followed by 5 mM ATP for 30 minutes. Data are presented as means ± SEM for three separate experiments, and each experiment was performed in triplicate. Cf-02, a benzamide-linked small molecule; 3-MA, 3-Methyladenine. ***P < .005. #Not detectable
FIGURE 8
FIGURE 8
Schematic representation of the plausible mechanism of action for therapeutic effects of Cf-02 on ASLN. ASLN, accelerated, severe lupus nephritis; Cf-02, a novel benzamide-linked small molecule
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References

    1. Tam LS, Li EK, Lai FM, Chan YK, Szeto CC. Mesangial lupus nephritis in Chinese is associated with a high rate of transformation to higher grade nephritis. Lupus. 2003;12:665–671. - PubMed
    1. Almaani S, Meara A, Rovin BH. Update on lupus nephritis. Clin J Am Soc Nephrol. 2017;12:825–835. - PMC - PubMed
    1. Weening JJ, D’Agati VD, Schwartz MM, et al. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int. 2004;65:521–530. - PubMed
    1. Kronbichler A, Kerschbaum J, Mayer G. The influence and role of microbial factors in autoimmune kidney diseases: a systematic review. J Immunol Res. 2015;2015:858027. - PMC - PubMed
    1. Whittall-Garcia LP, Torres-Ruiz J, Zentella-Dehesa A, et al. Neutrophil extracellular traps are a source of extracellular HMGB1 in lupus nephritis: associations with clinical and histopathological features. Lupus. 2019;28:1549–1557. - PubMed

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