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Novel approach to alleviate lupus nephritis: targeting the NLRP3 inflammasome in CD8+CD69+CD103+ TRM cells

Journal of Translational Medicine volume 22, Article number: 1139 (2024) Cite this article

Abstract

Background

Renal CD8+ tissue-resident memory T (TRM) cells display prolonged survival and activity in lupus nephritis (LN), exacerbating renal pathology. NLRP3 regulates the T cell response. This study explored the impact of NLRP3 inflammasome activity on the regulatory functions of TRM cells in LN.

Methods

NLRP3 inflammasome activity in renal CD8+ TRM cells from lupus-prone MRL/lpr mice and in vitro induced human CD8+CD103+ T cells was assessed by quantifying NLRP3, caspase-1, gasdermin D (GSDMD), and IL-1β levels using flow cytometry, ELISA, and western blotting analysis. The specific NLRP3 inhibitor MCC950, caspase-1 inhibitor Ac-YVAD-cmk, and NF-κB inhibitor JSH23 were utilized to delineate the role of NLRP3 in modulating the pathogenicity of CD8+ TRM cells in LN.

Results

Activation of the NLRP3 inflammasome was confirmed in renal CD8+CD69+CD103+ TRM cells derived from mice with LN and in vitro-induced human CD8+CD103+ TRM-like cells. MCC950 curtailed the infiltration and activity of CD8+CD69+CD103+ TRM cells and enhanced renal outcomes. MCC950 also suppressed the maturation and functional capabilities of CD8+CD103+ T cells in a manner reliant on inflammasome activity in vitro. IL-1β promoted the expression of TGF-βRII in CD8+ T cells via the NF-κB pathway.

Conclusions

NLRP3 inflammasome activity in renal CD8+CD69+CD103+ TRM cells contributes to LN pathogenesis by regulating cell differentiation and effector functions. Therapeutically targeting the NLRP3 inflammasome could significantly mitigate CD8+CD69+CD103+ TRM cell-mediated renal damage in LN.

Background

Systemic lupus erythematosus (SLE) represents a multifaceted and enduring autoimmune condition manifesting as imbalances of both innate and adaptive immune responses [1]. Lupus nephritis (LN), a critical manifestation of SLE, is characterized by the accumulation of immune complexes and immune cell infiltration in renal tissues [2]. Current therapeutic options for LN often yield suboptimal outcomes because of variable patient responses, recurrent disease episodes, and the adverse effects of treatments [3]. Therefore, it is important to deepen our understanding of the underlying mechanisms of LN to pinpoint new and more effective therapeutic approaches.

Among the immune cells implicated in LN, tissue-resident memory T (TRM) cells comprise a distinct subgroup of memory T cells situated in peripheral non-lymphoid tissues, and their robust effector capabilities have been clarified [4]. CD69 and CD103, instrumental in retaining TRM cells within peripheral tissues and preventing their egress, are commonly used as markers for TRM cells, with CD49a and CXCR3 being additional markers [5]. These TRM cells are associated with localized, recurrent inflammation responses, and they play roles in the flare-ups and progression of autoimmune conditions [6,7,8]. A notable elevation in the population of CD8+ TRM cells has been found within the renal environments of both patients with LN and lupus-prone mice [9, 10]. Renal CD8+ TRM cells continuously rejuvenate and exhibit robust effector functions that perpetuate inflammation and tissue damage in LN. Our previous studies have indicated that CD8+ TRM cells in LN kidneys are primarily CD69+CD103+ or CD69+CD103− [9]. Notably, among these, CD103+ cells exhibit enhanced functionality, suggesting they may occupy a more critical role in the pathogenesis of LN. Nonetheless, the precise mechanisms driving the pathogenic role of CD8+CD69+CD103+ TRM cells remain unclear.

NLRP3 inflammasome operates as a cytosolic assembly, being responsive to external and internal stimuli [11]. Its operational sequence begins with a priming phase that triggers transcriptional activation of its components, followed by inflammasome assembly across various pathways. Caspase-1 activation by this complex leads to the cleavage of gasdermin D (GSDMD), which forms pores facilitating the release of pro-inflammatory cytokines, including IL-1β and IL-18. Traditionally recognized as part of the innate immune system, NLRP3 influences adaptive immunity through IL-1β [12], and the importance of NLRP3 and IL-1β production within T cells themselves was recently highlighted [13,14,15,16,17,18,19,20]. The intrinsic activation of the NLRP3 inflammasome in CD4+ T cells leads to caspase-1-dependent IL-1β secretion, which promotes T helper 1 (Th1) cell differentiation and subsequently influences inflammatory processes in autoinflammatory diseases and infection models [13]. Conversely, in regulatory T (Treg) cells, NLRP3 impedes Foxp3 expression independently of inflammasome activation, thereby hindering Treg differentiation [18]. Additionally, the NLRP3 inflammasome, in conjunction with IRF4, acts as a transcription factor to mediate T helper 2 cell differentiation, independent of the inflammasome pathway [17]. Regarding CD8+ T cells, they exhibit increased NLRP3 and caspase-1 levels upon TCR and CD46 stimulation, without a corresponding increase in IL-1β in autoinflammatory diseases [15]. However, the study did not investigate specific cellular subsets or their roles in autoimmune diseases, indicating a gap that warrants further exploration.

Recent evidence has highlighted the NLRP3 inflammasome as a crucial mediator in the pathogenesis of various autoimmune diseases, including LN [21]. The NLRP3 inflammasome’s role in LN is still contested. Elevated expression of the NLRP3-IL-1β signaling axis in serum or renal tissues from lupus-prone mice has been demonstrated in our previous studies and is positively correlated with LN disease exacerbation [14, 22]. The inhibition of the NLRP3 inflammasome has been shown to alleviate lupus nephritis in lupus-prone mice [14, 22, 23]. However, the absence of NLRP3 led to the development of LN [24]. Thus, further elucidation is required to fully evaluate the activation of the renal NLRP3 inflammasome. Our researches demonstrated that NLRP3 inflammasome activity in T follicular helper (Tfh) cells and podocytes acts as a critical factor in the pathogenesis of LN by promoting germinal center formation and causing podocyte injury [14, 22]. However, the presence and impact of NLRP3 inflammasome activation within CD8+ T cells and its direct contribution to the progression of LN are not fully understood. Although the exact mechanisms of LN progression remain elusive, our current observations indicate that the NLRP3 inflammasome is active within renal CD8+CD69+CD103+ TRM cells in lupus-prone mouse models. Elevated autocrine IL-1β, mediated by NF-κB signaling, enhances TGF-βRII expression, augmenting CD8+ T cell susceptibility to TGF-β-driven differentiation and reinforcing a positive feedback loop that amplifies TRM cell pathogenicity. The findings support the concept that the NLRP3 inflammasome plays a significant role in the pathogenesis of CD8+CD69+CD103+ TRM cells in LN, suggesting that therapeutic strategies targeting NLRP3 inflammasome signaling hold potential in managing LN.

Methods

Patients

Patients meeting the American College of Rheumatology criteria for SLE with renal manifestations were recruited from the First Affiliated Hospital, Sun Yat-sen University [25]. Those with comorbid cancer, other autoimmune disorders, or infections were excluded from this study. Blood samples were obtained from LN patients who displayed symptoms of active disease, as quantified by an SLEDAI score greater than 6 [26]. This study received approval from the ethical review board of The First Affiliated Hospital, Sun Yat-sen University. All participants provided written informed consent. We also included age- and sex-matched healthy individuals as healthy controls (HCs).

Cell isolation

Blood samples were obtained from individuals with LN or HCs. Peripheral blood mononuclear cells (PBMCs) were isolated through centrifugation using Lymphoprep Density Gradient Medium (STEMCELL Technologies, Vancouver, CA) according to the product protocol instructions. Single-cell suspensions were prepared from mouse kidneys by following previously reported protocols [27]. The EasySep™ CD8+ T Cell Isolation Kit (STEMCELL Technologies) was applied for the enrichment of human CD8+ T cells, ensuring that cell purity exceeded 95%, as verified by flow cytometry (> 95%). The CD103+ and CD103− subsets of CD8+ T cells were isolated using flow cytometry on a FACSAria system (BD Biosciences, Franklin Lakes, NJ, USA).

Cell culture

We activated the isolated human CD8+ T cells using Human T-Activator CD3/CD28 Dynabeads (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at a ratio of 1:4 (beads: cells) within basal RPMI 1640 medium containing 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cells were incubated at 37 °C in a 5% CO2 environment. For human CD8+ T cell differentiation, the cells were cultured in complete medium containing 10 ng/ml TGF-β (PeproTech, Cranbury, NJ, USA) for 3 days. Various concentrations of MCC950 (ranging from 1 to 20 µM; Selleck Chemicals, Houston, TX, USA), along with Ac-YVAD-cmk (ranging from 1 to 40 µM; Selleck Chemicals), IL-1β (10 ng/ml; PeproTech), IL-15 (10 ng/ml; PeproTech), IL-18 (10 ng/ml; PeproTech), IL-1 receptor antagonist (20 µg/ml; MedChemExpress, Monmouth Junction, NJ, USA), a human TGF-βR II antibody (10 µg/mL; R&D systems, Minneapolis, MN, USA) and the NF-κB inhibitor JSH23 (30µM; MedChemExpress) were included in specific experimental setups.

Quantitative real-time PCR (qPCR)

qPCR was employed to detect gene expression. First, total RNA was extracted from cells using the RNA Quick Purification kit (ESscience, Shanghai, China). cDNA was subsequently generated using the Evo M-MLV RT Premix (Accurate Biology, Hunan, China). qPCR was conducted using the SYBR Green qPCR Kit (Accurate Biology). The primer sequences used in the amplification of human NLRP3, caspase-1, IL-1β, IL-18, and β-actin were as follows: NLRP3, 5′-GACCCAGGGATGAGAGTGTTGTG-3′ (forward) and 5′- GCTGCTGAGGACCAAGGAGATG-3′ (reverse); caspase-1, 5′- ATCTCACTGCTTCGGACATGACTAC-3′ (forward) and 5′- GTTTCTTCCCACAAATGCCTTCCC-3′ (reverse); IL-1β, 5′-CAGTGGCAATGAGGATGACTTGTTC-3′ (forward) and 5′- CTGTAGTGGTGGTCGGAGATTCG-3′ (reverse); IL-18, 5′- GGCTGCTGAACCAGTAGAAGAC-3′ (forward) and 5′- AGCTTGCCAAAGTAATCTGATTCC-3′ (reverse); β-actin, 5′- CCTGGACTTCGAGCAAGAGATGG-3′ (forward) and 5′- CAGGAAGGAAGGCTGGAAGAGTG-3′ (reverse). For amplification, cDNA was subject to denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. The levels of the detected genes were then normalized to β-actin, and the results were quantified as relative expression using a qPCR system (Applied Biosystems, Thermo Fisher Scientific).

Western blotting

Human CD8+ T cells were lysed in RIPA buffer, with approximately one hundred thousand cells isolated per sample. The protein concentration of the lysate was detected using a BCA Protein Assay Kit (Thermo Fisher Scientific; Cat#: 23227). For each lane, 50 µg of protein was loaded onto a 10% or 15% SDS-PAGE gel. The gels, measuring 81 × 74 × 1.5 mm, were prepared using the PAGE Gel Fast Preparation Kit (Epizyme Biomedical Technology, Shanghai, China; Cat#: PG112 and PG114). Protein separation was performed using a Tanon system (Tanon, Shanghai, China; Cat# EPS300). After separation, the proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Tris-Glycine SDS-PAGE Running Buffer was used for both the stacking and resolving stages, while Tris-Glycine Transfer Buffer was employed for the transfer process. The membranes were subsequently blocked with 5% bovine serum albumin in TBST buffer. Following this, the membranes were incubated with antibodies specific for caspase-1 p20 (AdipoGen, San Diego, CA, USA; Cat#: AG-20B-0048), NLRP3 (AdipoGen; Cat#: AG-20B-0014), GSDMD (Cell Signaling Technology, Danvers, MA, USA; Cat#: 39754S), GAPDH (Cell Signaling Technology; Cat#: 2118S), phospho-TAK1 (Cell Signaling Technology; Cat#: 9339S), TAK1 (Cell Signaling Technology; Cat#: 5206S), phospho-IKKα/β (Cell Signaling Technology; Cat#: 2697S), IKKα (Cell Signaling Technology; Cat#: 11930S), IKKβ (Cell Signaling Technology; Cat#: 8943S), phospho-IKBα (Cell Signaling Technology; Cat#: 2859S), IKBα (Cell Signaling Technology; Cat#: 4814S), phospho-NF-κB p65 (Cell Signaling Technology; Cat#: 3033S), and NF-κB p65 (Cell Signaling Technology; Cat#: 8242S) at 4 °C overnight. After three rinses with TBST buffer, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse (Cat#: 7076S) or anti-rabbit (Cat#: 7074S) antibody (Cell Signaling Technology). Signal detection was performed by enhanced chemiluminescence using a Chemilucent Plus Western Blot Enhancing Kit (MilliporeSigma, Burlington, MA, USA).

Coomassie brilliant blue staining

Proteins were visualized following electrophoresis using Coomassie Brilliant Blue Rapid Staining Solution (Servicebio Technology, Wuhan, China; Cat#: G2059). Initially, the gels were rinsed with deionized water three times for 20 s each. They were then stained in the staining solution with shaking for 30 min, followed by destaining in deionized water for 30 min. The stained gel was photographed using the G: BOX EF gel imaging system (Syngene, Cambridge, UK).

Enzyme-linked immunosorbent assay (ELISA)

ELISA was utilized to quantify IL-1β levels in mouse serum, renal tissue homogenates and human in vitro cell culture supernatants, using an ELISA kit (Cusabio, Wuhan, China; Cat#: CSB-E08053h), according to the manufacturer’s protocol. For the cell culture supernatants, human CD8+ T cells were initially cultured with anti-CD3/CD28 Dynabeads in the presence of TGF-β for three days. Subsequently, the cells were sorted into CD103− and CD103+ populations and cultured for an additional three days in TGF-β-free conditions. The culture supernatant was collected and underwent ELISA analysis.

Mouse experiments

Female MRL/lpr mice were obtained from the SLAC Laboratory Animal Center (Shanghai, China). C57B6/L mice, age- and gender-matched, were acquired from the Experimental Animal Center at Guangzhou University of Chinese Medicine (Guangzhou, China). The mice were housed under specific pathogen-free conditions in the Experimental Animal Center at Sun Yat-sen University. The mouse experimental protocol was approved by the Ethics Committee of Sun Yat-sen University. All procedures were conducted following the guidelines of the National Institutes of Health Guide for Care and Use of Animals. Mice were administered MCC950 (10 mg/kg) or vehicle (5% DMSO, 40% PEG300, and 5% Tween 80 in water) via daily intraperitoneal injections from 12 to 18 weeks of age. Urine samples were weekly collected to monitor protein levels. At the end of the treatment period, the mice were euthanized, and their kidneys were harvested for tissue staining and single-cell analysis.

Flow cytometry

For surface marker and FAM-FLICA caspase-1 detection, cells were labeled with antibodies targeting APC/cyanine 7 anti-human CD8 (Cat#: 344714), PE/cyanine 7 anti-human CD103 (Cat#: 350212), PE anti-human CD49a (Cat#: 328303), Brilliant Violet 510 anti-human CXCR3 (Cat#: 353726), eFluor 660 anti-human S1PR1 (Thermo Fisher Scientific; Cat#: 50-3639-41), PE anti-human CD62L (Cat#: 385103), APC anti-human TGF-βRII(Cat#: 399706), APC anti-mouse CD45 (Cat#: 103112), Alexa Fluor 700 anti-mouse CD45 (Cat#: 103128), Brilliant Violet 650 anti-mouse CD3 (Cat#: 100229), APC/Fire 750 anti-mouse CD4 (Cat#: 100568), PE/cyanine 7 anti-mouse CD8 (Cat#: 100722), Brilliant Violet 510 anti-mouse CD8 (Cat#: 100752), PE anti-mouse CD19 (Cat#: 115507), PE/cyanine 7 anti-mouse CD49a (Cat#: 142607), PE/cyanine 7 anti-mouse CXCR3 (Cat#: 126515), FITC anti-mouse CD69 (Cat#: 104505), APC anti-mouse CD69 (Cat#: 104514), Brilliant Violet 421 anti-mouse CD103 (Cat#: 121422), and a FAM-FLICA caspase-1 probe (ImmunoChemistry Technologies, Davis, CA, USA; Cat#: 98) at 4℃ for 30 min. Then, cells were rinsed twice with PBS. For intracellular staining, cells were initially treated with the BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences) and stained with the corresponding antibodies including APC anti-human perforin (Cat#: 353312), PE anti-mouse perforin (Cat#: 154306), PE anti-human/mouse granzyme B (GZMB) (Cat#: 372208), PE/cyanine 7 anti-human/mouse GZMB (Cat#: 372214), and BV421 anti-mouse CD68 (Cat#: 137017). For NLRP3 staining, cells were incubated with a primary antibody against NLRP3 (1:50; Abcam; Cat#: ab263899), followed by incubation with a PE-conjugated Donkey anti-Rabbit IgG secondary antibody (1:50; Thermo Fisher Scientific; Cat#: 12-4739-81). To stain intracellular cytokines, the cells were treated with 500 ng/ml ionomycin (Sigma-Aldrich, St. Louis, MO, USA; Cat#: I0634), 50 ng/ml PMA (Sigma-Aldrich; Cat#: P8139), and 5 µg/ml brefeldin A (Sigma-Aldrich; Cat#: B7651) for 5 h, fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences), and reacted with APC anti-human TNFα (Cat#: 502912), Pacific Blue anti-human IL-1β (Cat#: 511709), PE anti-human IFNγ (Cat#: 502509), PE/cyanine 7 anti-human TGF-β (Cat#: 300007), PE/cyanine 7 anti-mouse IL-1β (Thermo Fisher Scientific; Cat#: 25-7114-80), Brilliant Violet 605 anti-mouse IFNγ (Cat#: 505840), PE/cyanine 7 anti-mouse TNFα (Cat#: 506324), and PE anti-mouse TGF-β (Cat#: 141306) at 4℃ for 30 min. The surface antibody-labeled cells were further fixed and permeabilized using a Foxp3 Staining Set (eBioscience, San Diego, CA, USA) and reacted with Brilliant Violet 510 anti-human ki-67 (Cat#: 350518), and APC anti-mouse ki-67 (Cat#: 652406) at 4℃ for 30 min for Ki-67 staining. For the analysis of cellular apoptosis, the cells were stained with annexin V (Cat#: 640907) and 7-AAD (Cat#: 420403). All antibodies applied in this study were purchased from BioLegend (San Diego, CA, USA) unless indicated otherwise. Samples were examined using a CytoFLEX S Flow Cytometer (Beckman Coulter, Brea, CA, USA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Detection of proteinuria, serum creatinine and blood urea nitrogen (BUN) levels

Mouse urine specimens were evaluated using Siemens Albustix Reagent Strips (Siemens Healthineers, Munich, Germany). Six distinct color bands were scored as follows: 0, negative or trace protein; 1, 30–100 mg/dl protein; 2, 100–300 mg/dl protein; 3, 300–2000 mg/dl protein; and 4, ≥ 2000 mg/dl protein. Severe proteinuria was designated for samples with protein levels ≥ 300 mg/dl. Renal function was evaluated by detecting the serum concentrations of creatinine and BUN using a blood biochemistry autoanalyzer (Beckman Coulter).

Evaluation of glomerulonephritis

Mouse kidney samples were preserved in 4% polyoxymethylene, dried, imbedded in paraffin, and sectioned at a thickness of 2 μm. The sections were subsequently stained using periodic acid-Schiff (PAS). Histopathologic assessments were performed by two independent, blinded observers as previously reported [28]. The severity of glomerular and vascular lesions was scored on a scale of 0–3.

Statistical analysis

All data are presented as the mean ± SEM. Statistical evaluations were conducted via GraphPad Prism 9.0 (GraphPad, La Jolla, CA, USA). The t-test, one-way ANOVA or two-way ANOVA was applied to detect differences between the groups. Statistical significance was defined as p < 0.05 in two-tailed tests.

Results

NLRP3 inflammasome activation in CD8+CD69+CD103+ TRM cells

To explore whether the NLRP3 inflammasome is activated in renal CD8+ TRM cells in LN, the expression of active caspase-1, NLRP3 and IL-1β was measured in CD8+ T cells isolated from 18-week-old MRL/lpr mice, model of LN, and compared with those from normal control C57BL/6 mice using flow cytometry (Fig. 1A). Figure 1B presents a comprehensive overview of the gating strategy, CD69−CD103−, CD69+CD103− and CD69+CD103+ cells were identified within the CD8+ T cell population. Both the CD8+CD69+CD103− and CD8+CD69+CD103+ cell subsets express the TRM cell markers CD49a and CXCR3, with significantly lower expression observed in CD8+CD69−CD103− cells (Figure S1A–S1D). A FAM-FLICA caspase-1 probe was employed to detect the level of active caspase-1, a biomarker of NLRP3 inflammasome activation. Comparable levels of active caspase-1 were observed among the CD8+CD69−CD103−, CD8+CD69+CD103−, and CD8+CD69+CD103+ cell subsets in the renal tissues of C57 mice, as well as between the CD8+CD69−CD103− and CD8+CD69+CD103− populations in MRL/lpr mice (Fig. 1C and D). However, the CD8+CD69+CD103+ subset in MRL/lpr mice demonstrated higher active caspase-1 expression than the other two subsets and also had increased levels compared to CD8+CD69+CD103+ cells in C57 kidneys (Fig. 1C and D). Analogous patterns were noted in the levels of NLRP3 and IL-1β (Fig. 1E and H). IL-1β levels in the serum of MRL/lpr mice were higher compared to those in C57BL/6 mice (Fig. 1I), and this pattern was also observed in kidney tissues (Fig. 1J).

Fig. 1
figure 1

Enhanced NLRP3 inflammasome activation in renal CD8+CD69+CD103+ TRM cells of lupus-prone mice. A: Eighteen-week-old C57BL/6 and MRL/lpr mice were euthanized, serum and kidneys were collected for further analysis. B: Gating strategy. Single-cell suspensions were isolated from kidneys. Flow cytometry was used to analyze the cells. Dead cells were initially excluded by zombie staining. CD45+ cells were selected and further gated on CD3+CD8+ T cells. CD3+CD8+ T cells were then sorted into CD69−CD103−, CD69+CD103−, and CD69+CD103+ cell populations for further analysis. C–H: The expression of active caspase-1 (C and D), NLRP3 (E and F) and IL-1β (G and H) was assessed by flow cytometry. Representative plots focusing on renal CD8+CD69−CD103−, CD8+CD69+CD103− and CD8+CD69+CD103+ cells are presented. I–J: ELISA analysis was conducted on the collected serum (I) and renal homogenates (J). Data are presented as mean ± SEM from five mice. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA corrected for multiple comparisons (D–H) and unpaired Student’s t-test (I and J). ns, not significant

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We further investigated the relationship between the NLRP3 signaling pathway and CD8+CD103+ TRM-like cells in vitro. Initially, we endeavored to establish an in vitro induction model for CD8+CD103+ TRM-like cells. TGF-β signaling is absolutely required for CD8+CD103+ TRM cell induction and persistence [9, 29, 30], whereas IL-15 is known to support the activation and survival of TRM cells [31]. To ascertain whether TGF-β1 alone can elicit a TRM-like phenotype, CD8+ T cells from PBMCs of HCs, induced by TGF-β1 alone or in combination with IL-15, were analyzed for CD103+ proportions, activity levels, and TRM-associated characteristics (Figure S2A). Figures S2B and S2C demonstrate that both treatments significantly induced a high proportion of CD103+ cells, with no significant differences observed between the two groups. There were no notable differences in Annexin V, 7-AAD, and Ki-67 expression between the two groups, implying comparable cellular vitality and proliferation potential (Figure S2D–S2G). As shown in Figure S2H and S2I, the CD103+ cells exhibited upregulation of CD49a and CXCR3, and downregulation of S1PR1 and CD62L, when compared to CD103− cells, which is consistent with the characteristics of TRM-like cells. Notably, significant differences were not observed between the two induction groups. Incorporating these results, it was found that TGF-β effectively induces the generation of TRM-like cells in vitro.

Further analysis was conducted to assess the activation of the NLRP3 inflammasome pathway in CD8+CD103+ TRM-like cells induced by TGF-β1 in vitro (Fig. 2A). The increased levels of active caspase-1 and IL-1β in CD8+CD103+ T cells were confirmed by flow cytometry (Fig. 2B and E). We then sorted CD8+ T cells into CD103− and CD103+ subsets using flow cytometry (Fig. 2F). The mRNA levels of NLRP3, caspase-1, and IL-1β were obviously higher in CD8+CD103+ cells than in CD8+CD103− cells (Fig. 2G). Although IL-18 mRNA levels were higher in CD8+CD103+ cells, the difference was not significant (Fig. 2G). In addition, CD8+CD103+ cells exhibited greater expression of NLRP3 and caspase-1 p20, a marker of active caspase-1, as demonstrated by western blotting (Fig. 2H and I). Coomassie Brilliant Blue staining also verified the uniformity of protein amounts loaded across different lanes (Figure S3A). Elevated levels of cleaved GSDMD, indicative of NLRP3 inflammasome activation, were also detected in CD8+CD103+ cells, as revealed by western blotting (Fig. 2J and K). Moreover, ELISA data indicated that CD103+ cells secreted greater quantities of IL-1β than their CD103− counterparts (Fig. 2L). We then evaluated the proliferative capacity and effector functions of these two subsets. As presented in Figure S4A and S4B, CD8+CD103+ T cells exhibited higher Ki-67 levels than CD8+CD103− T cells, suggesting stronger cellular proliferation. Furthermore, higher percentages of perforin-, GZMB-, TNFα-, and IFNγ-positive cells were observed in the CD8+CD103+ cell subset (Figure S4C–S4I). The hyperactive status of CD8+CD103+ TRM-like cells was consistent with our previous results in kidneys from lupus-prone mice [9]. Overall, activation of the NLRP3 inflammasome in CD8+CD103+ TRM cells is significantly associated with their developmental and functional properties, potentially offering substantial insights into the biology of CD8+CD103+ TRM cells.

Fig. 2
figure 2

Enhanced NLRP3 inflammasome activation in human CD8+CD103+ TRM-like cells. A–E: The isolated CD8+ T cells from the PBMCs of HCs were treated with anti-CD3/CD28 Dynabeads and 10 ng/ml TGF-β for 3 days to induce CD103 expression. A: Experiment scheme. B–E: Active caspase-1 (B and C) and IL-1β (D and E) levels were determined by flow cytometry. Representative dot plots are presented, concentrating on CD8+CD103− and CD8+CD103+ cell populations. F–L: CD8+ T cells isolated from the PBMCs of HCs were treated with TGF-β for 3 days and then sorted into CD8+CD103− and CD8+CD103+ populations for qPCR, western blotting and further ELISA analysis. F: Experiment scheme. G: RNA was extracted from CD8+CD103− and CD8+CD103+ cells, and the transcripts of NLRP3, caspase-1, IL-1β, and IL-18 were examined by qPCR (n = 3). H–K: CD8+CD103− and CD8+CD103+ cells were subjected to western blotting to detect the expression of NLRP3, pro caspase-1, active caspase-1 p20, full length GSDMD, cleaved GSDMD, and GAPDH. Representative bands and their densities are presented (n = 3). L: Sorted CD8+CD103− and CD8+CD103+ cell populations were further cultured for an additional three days with anti-CD3/CD28 Dynabeads in the absence of TGF-β. The culture supernatant was then harvested, and IL-1β levels were assessed by ELISA (n = 3). Data are displayed as the mean ± SEM. *p < 0.05, and ****p < 0.0001, paired Student’s t-test. ns, not significant

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CD8+ T cells from patients with LN had a greater propensity to transform into CD8+CD103+ cells, exhibiting higher levels of NLRP3 inflammasome activation

To further analyze the role of NLRP3 inflammasome activation in the differentiation of CD8+CD103+ TRM-like cells within patients with LN, circulating CD8+ T cells from both HCs and patients with LN were exposed to TGF-β. CD8+ T cells from patients with LN displayed a higher likelihood of transforming into CD8+CD103+ cells than those from HCs (Fig. 3A and B), as previously reported [9]. This suggests that circulating CD8+ T cells from patients with LN have a higher propensity to transform into TRM-like cells once they migrate to the kidneys. It is noteworthy that CD8+CD103+ cells from patients with LN displayed higher levels of active caspase-1 than those from HCs (Fig. 3C and D). Moreover, NLRP3 and caspase-1 p20 were upregulated in CD8+CD103+ cells from patients with LN compared with their expression in HCs, as assessed by western blotting (Fig. 3E and F). The uniformity of protein loading was also ascertained using Coomassie Brilliant Blue staining (Figure S3B). CD8+CD103+ cells also exhibited increased levels of cleaved GSDMD, as determined by western blotting (Fig. 3G and H). IL-1β secretion by CD8+CD103+ cells from patients with lupus nephritis was found to be elevated relative to those derived from HCs, as determined by ELISA (Fig. 3I). These findings highlight a significant association between the formation of CD8+CD103+ TRM-like cells and NLRP3 inflammasome activation in LN.

Fig. 3
figure 3

CD8+ T cells from LN patients displayed a greater tendency to differentiate into CD8+CD103+ cells. CD8+ T cells isolated from the PBMCs of patients with LN or HCs were triggered by anti-CD3/CD28 Dynabeads and 10 ng/ml TGF-β for 3 days to induce CD103+ cell differentiation. A and B: CD103 expression on CD8+ T cells was assessed by flow cytometry. Representative contour plots and percentages of CD8+CD103+ T cells are displayed (n = 5). C and D: Activated caspase-1 levels were quantified by flow cytometry, and representative images concentrating on CD8+CD103+ T cells are presented (n = 5). E–I: Following 3 days of stimulation, CD8+CD103+ cells from patients with LN or HCs were sorted. E–H: Cells were lysed and analyzed for NLRP3, pro caspase-1, caspase-1 p20, full length GSDMD, cleaved GSDMD, and GAPDH by western blotting. Representative bands and their densities are presented (n = 3). I: Sorted CD8+CD103+ cells were further cultured for an additional three days with anti-CD3/CD28 Dynabeads in the absence of TGF-β. The culture supernatant was then harvested, and IL-1β levels were assessed by ELISA (n = 3). Data are displayed as the mean ± SEM. *p < 0.05, **p < 0.01, and ****p < 0.0001, unpaired Student’s t-test

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MCC950 inhibited NLRP3 inflammasome activation and reduced renal lesions in lupus-prone MRL/lpr mice

To ascertain the role of the NLRP3 inflammasome in CD8+ TRM cells in LN, 12-week-old MRL/lpr mice were administered MCC950, a specific NLRP3 inhibitor, for 6 weeks (Fig. 4A). MCC950 treatment markedly decreased active caspase-1 levels in renal CD8+CD69+CD103+ TRM cells (p < 0.0001), yet had no impact on levels in CD8+CD69+CD103− TRM cells (Fig. 4B and D). MCC950 treatment also effectively downregulated the expression of NLRP3 and IL-1β in CD8+CD69+CD103+ TRM cells (Fig. 4E and H) and reduced IL-1β expression in the serum and kidneys (Fig. 4I and J). Consistent with our previous study [14], MCC950 treatment reduced proteinuria, blood BUN and creatinine levels, glomerular damage, and perivascular cellular infiltration (Fig. 4K and O).

Fig. 4
figure 4

MCC950 treatment alleviated renal lesions in lupus-prone mice. MRL/lpr mice were administered 10 mg/kg MCC950 or vehicle starting at 12 weeks of age for 6 weeks. Serum and kidneys were then collected. A: Experimental design of the mouse study. B–D: Single-cell suspensions of mouse kidneys were prepared. The expression of active caspase-1 was assessed by flow cytometry. Representative plots focusing on CD8+CD69+CD103− and CD8+CD69+CD103+ cells are presented. Data were collected from eight mice. E–H: Single-cell suspensions of mouse kidneys were prepared. The expression of NLRP3 (E and F) and IL-1β (G and H) was assessed by flow cytometry. Representative plots focusing on CD8+CD69+CD103+ cells are presented. Data were collected from five mice. I and J: ELISA analysis was conducted on the collected serum (I) and renal homogenates (J). Data were collected from five mice. K: The proteinuria score was recorded in mice treated with either MCC950 or vehicle. Data were collected from eight mice. L and M: Serum levels of BUN and creatinine from treated mice were analyzed using a blood biochemistry auto-analyzer. Data were collected from eight mice. N and O: Sections of renal samples were stained with PAS for pathological analysis. Typical images and histopathological scores were recorded at an original magnification of ×400. Data were collected from eight mice. All data are displayed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 by unpaired Student’s t-test. ns, not significant

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It is noted that MCC950 selectively inhibited NLRP3 inflammasome activation in renal CD8+CD69+CD103+ TRM cells. A significant reduction in active caspase-1 expression within renal CD8+ T cells was observed in the MCC950-treated group (p < 0.01, Figures S5, S6A and S6B), primarily attributable to the presence of CD8+CD69+CD103+ TRM cells. However, MCC950 did not markedly affect caspase-1 activation in renal CD4+ T cells, CD3+CD4−CD8− double-negative T (DNT) cells, or B cells (Figures S5, S6C–S6H). MCC950 slightly decreased active caspase-1 expression in macrophages (p = 0.043, Figures S5, S6I and S6J), which might not have biological relevance. Collectively, in the case of local immune cells, NLRP3 inflammasome activation might contribute to the pathogenesis of LN mainly through regulating CD8+CD69+CD103+ TRM cells, but not other immune cells.

MCC950 treatment suppressed CD8+ TRM cell development and effector functions

To further investigate whether MCC950 influences the development and functional status of CD8+CD69+CD103+ TRM cells in vivo, we utilized flow cytometry to assess the expression of Ki-67 and representative effector molecules. MCC950 treatment significantly reduced the number of renal CD8+CD69+CD103+ TRM cells and proportion of CD8+CD69+CD103+ TRM cells within CD8+ cells (Fig. 5A and C), suggesting a negative impact of MCC950 on CD8+CD69+CD103+ TRM cell differentiation. Renal CD8+CD69+CD103+ TRM cells displayed markedly decreased Ki-67 expression following MCC950 treatment (Fig. 5D and E). Additionally, CD8+CD69+CD103+ TRM cells from the kidneys of MCC950-treated mice exhibited significantly decreased expression of perforin and GZMB in comparison with those from vehicle-treated mice (Fig. 5F and I). MCC950 treatment also reduced the synthesis of IFNγ and TNFα in renal CD8+CD69+CD103+ TRM cells (Fig. 5J and L). These findings revealed that NLRP3 is crucial for the maintenance, development, and effector functions of CD8+CD69+CD103+ TRM cells in the kidneys of lupus-prone MRL/lpr mice.

Fig. 5
figure 5

MCC950 treatment suppressed CD8+CD69+CD103+ TRM cell formation and effector function in lupus-prone mice. MRL/lpr mice were treated with 10 mg/kg MCC950 or vehicle starting at 12 weeks of age for 6 weeks. Kidneys were collected to prepare single-cell suspensions. A–C: Flow cytometry was performed and representative plots were gated on CD8+ cells. Panel B displays the absolute numbers of CD8+CD69+CD103+ T cells in one kidney from mice treated with MCC950 or vehicle. Panel C presents the proportion of CD69+CD103+ cells in the CD8+ T cell population in the kidneys of mice treated with either MCC950 or vehicle. Data were collected from eight mice. D–L: Expression of Ki-67 (D and E), perforin (F and G), GZMB (H and I), TNFα and IFNγ (J–L)was analyzed by flow cytometry. Representative plots focusing on CD8+CD69+CD103+ cells are presented. All data are presented as the mean ± SEM. ****p < 0.0001, unpaired Student’s t-test

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MCC950 treatment inhibited the differentiation and functionality of human CD8+CD103+ TRM-like cells in an inflammasome-dependent manner

To verify the specific activity of the NLRP3 inflammasome in the differentiation and activity of CD8+CD103+ TRM cells, circulating CD8+ T cells from HCs were treated with TGF-β in vitro to induce TRM-like phenotypes. The data demonstrated that 10 µM MCC950 effectively prevented the differentiation of CD8+ cells into CD8+CD103+ TRM-like cells (Figures S7A, S7B, 6 A, and 6B), suggesting that NLRP3 contributed to CD8+ TRM cell development. Ac-YVAD-cmk, a specific caspase-1 inhibitor, was applied to suppress caspase-1 activity to confirm the role of the NLRP3 inflammasome in shaping the CD8+CD103+ TRM status. Similarly, Ac-YVAD-cmk treatment inhibited CD8+CD103+ differentiation (Figures S7C, S7D, 6 A, and 6B), suggesting that NLRP3 inhibitor suppressed TRM differentiation through an inflammasome-dependent mechanism. Moreover, flow cytometry confirmed that both MCC950 and Ac-YVAD-cmk effectively inhibited caspase-1 activation, as evidenced by a decreased mean fluorescence intensity (MFI) for active caspase-1 (Fig. 6C and D). The effects of NLRP3 inhibition on CD103+ TRM-like cell proliferation capacity and functions were further examined. As presented in Fig. 6E and F, Ki-67 expression on CD8+ TRM-like cells was significantly suppressed by MCC950 treatment. In addition, MCC950 treatment significantly suppressed their functions, as evidenced by decreased MFIs for perforin and GZMB and lower production of TNFα and IFNγ (Fig. 6G and L). Similar results were observed using Ac-YVAD-cmk treatment (Fig. 6E and L). These data suggested that NLRP3 inflammasome signaling is necessary for the differentiation, cellular proliferation and effector functions of CD8+CD103+ T cells.

Fig. 6
figure 6

MCC950 treatment inhibited the differentiation and activity of human CD8+CD103+ cells in an inflammasome-dependent manner. CD8+ T cells isolated from the PBMCs of HCs were treated with anti-CD3/CD28 Dynabeads and 10 ng/ml TGF-β for 3 days to induce CD103+ cell differentiation. Some experiments included MCC950 (10 µM), Ac-YVAD-fmk (10 µM). A and B: CD103 expression was detected by flow cytometry, concentrating on CD8+ cells. C and D: Active caspase-1 expression was analyzed by flow cytometry. Representative plots focusing on CD8+CD103+ cells are presented. E–M: Ki-67 (E and F), perforin (G and H), GZMB (I and J), TNFα and IFNγ (K and L) expression was analyzed by flow cytometry, concentrating on CD8+CD103+ cells. All data are presented as the mean ± SEM of five independent assays. **p < 0.01, ***p < 0.001, and ****p < 0.0001, one-way ANOVA corrected for multiple comparisons. ns, not significant

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A recent study has revealed that TGF-β supports epidermal CD8+ TRM cells through an autocrine mechanism [32]. Interestingly, NLRP3 inflammasome signaling has been found to promote TGF-β expression in several tissues [33, 34]. We then investigated whether renal CD8+ TRM cells produce TGF-β. As illustrated in Figure S8A and S8B, renal CD8+CD69+CD103+ TRM cells from 18-week-old diseased MRL/lpr mice displayed higher TGF-β expression than CD8+CD69+CD103− TRM or CD8+CD69−CD103− non-TRM cells. In vitro, circulating CD8+ T cells from HCs were isolated and cultured under TGF-β stimulation. Enhanced expression of TGF-β was also found in induced human CD8+CD103+ TRM−like cells (Figure S8C and S8D). No difference in TGF-β expression was found between stimulated CD8+CD103− T cells and unstimulated CD8+ T cells. Interestingly, MCC950 treatment significantly decreased intrinsic TGF-β expression in renal CD8+CD69+CD103+ TRM cells from MRL/lpr mice (Figure S8E and S8F). Meanwhile, anakinra, an IL-1β receptor antagonist, inhibited TGF-β production in induced CD8+CD103+ TRM−like cells in vitro (Figure S8G and S8H). These data collectively indicated that NLRP3 promotes endogenous TGF-β production in CD8+CD103+ TRM cells in an inflammasome-dependent manner, which might contribute to the differentiation and maintenance of TRM cells.

IL-1β promoted TGF-βRII expression in human CD8+ T cells via NF-κB signaling

It is well known that caspase-1 activation is triggered by the NLRP3 inflammasome, resulting in IL-1β and IL-18 activation [11]. CD8+ T cells, derived from PBMCs of HCs, were subjected to TGF-β stimulation and subsequently differentiated into CD8+CD103+ TRM−like cells. We discovered that IL-1β and IL-18 partially reversed the reduction in the proportion of CD103⁺CD8⁺ TRM−like cells following MCC950 treatment (Fig. 7A and B), which further supporting the hypothesis that NLRP3 is involved in CD8+CD103+ TRM cell development in an inflammasome-dependent manner. Subsequent studies were undertaken to evaluate the effects of IL-1β and IL-18 on the induction of CD8+CD103+ TRM−like cells. Our results indicated that IL-1β alone was not sufficient to induce differentiation of CD8⁺CD103⁺ TRM−like cells; however, its addition to TGF-β1 significantly enhanced their induction (Fig. 7C). In contrast, IL-18 did not appear to influence the differentiation of CD8⁺CD103⁺ cells. It is known that TGF-β signals through TGF-βRII to induce and sustain TRM cells [30]. Interestingly, neutralization of TGF-βRII abrogated the induction effect of TGF-β1 plus IL-1β on CD8⁺CD103⁺ cells (Fig. 7C), suggesting that IL-1β’s potentiating role in TGF-β-mediated TRM−like cell induction may be associated with TGF-βRII. IL-1β is known to activate the NF-κB pathway, which is crucial for the regulation of inflammasome-dependent cellular responses [35]. The addition of IL-1β to TGF-β1 significantly enhanced TAK1/NF-κB pathway activation in CD8+ T cells, as evidenced by increased phosphorylation of TAK1, IKKα/β, IκBα, and NF-κB p65 (Fig. 7D and H), along with elevated TGF-βRII expression (Fig. 7I and J). This upregulation of TGF-β RII expression was substantially reversed by NF-κB pathway inhibition with JSH23 (Fig. 7I and J). The above results suggest that IL-1β may promote the development of CD103⁺CD8⁺ TRM cells by enhancing TGF-βRII expression via the TAK1/NF-κB pathway.

Fig. 7
figure 7

IL-1β promoted the expression of TGF-βRII via NF-κB signaling. The isolated CD8+ T cells from the PBMCs of HCs were treated with anti-CD3/CD28 Dynabeads for 3 days, in the presence of 10 ng/ml TGF-β. MCC950 (10 µM), IL-1β (10 ng/ml), IL-18 (10 ng/ml), anti-TGF-βRII antibody (10 µg/mL) and JSH-23 (30 µM) were included in some experiments. A and B: CD103 expression was detected by flow cytometry, with representative plots focusing on CD8+ cells (n = 5). C: CD103 expression was detected by flow cytometry. Percentages of CD103+ cells gated on CD8+ T cells were shown (n = 3). D–H: Cells were subjected to western blotting to detect the expression of p-TAK1, TAK1, p-IKKα/β, IKKα, IKKβ, p-IKBα, IKBα, p-p65, p65 and GAPDH. Representative bands and their densities are presented (n = 3). I and J: TGF-βRII levels were determined by flow cytometry. Representative plots are presented, concentrating on CD8+ cell populations (n = 3). *p < 0.05, **p < 0.01, ****p < 0.0001, one-way ANOVA corrected for multiple comparisons (B, C and J) and unpaired Student’s t-test (E–H). ns, not significant

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Discussion

CD8+ TRM cells play vital roles in local inflammation, driving the progression and recurrence of autoimmune disease [36]. Our previous study revealed that the CD8+ TRM cell population was markedly increased following LN establishment in rodents. These cells maintained self-renewal and exhibited strong functions, persistently promoting renal inflammation and injury in LN [9]. In addition, recently Li et al. found that CD8+ TRM cells triggered podocyte injury and aggravated glomerulosclerosis [37]. Studies have confirmed that a combination of extracellular signals (e.g., TGF-β and IL-15) and intrinsic mechanisms (e.g., signaling downregulation of KLF2 and T-bet) promote T cell commitment to the TRM phenotype and the corresponding behaviors [10, 37, 38]. However, CD8+ TRM cells exhibit interorgan heterogeneity, and the precise mechanism regulating CD8+ TRM cells in LN remains unclear. Our study confirmed hyperactivation of the NLRP3 inflammasome in renal CD8+CD69+CD103+ TRM cells, rather than in CD8+CD69+CD103− TRM cells or CD8+CD69−CD103− non-TRM cells, in lupus-prone MRL/lpr mice. This hyperactivation was effectively inhibited by the NLRP3 inhibitor MCC950, resulting in significant suppression of renal CD8+CD69+CD103+ TRM cells and improvement of renal pathology. The role of the NLRP3 inflammasome in the development and activity of CD8+CD103+ TRM cells was also confirmed using in vitro induced CD8+CD103+ TRM-like cells. NLRP3 inflammasome modulates LN inflammation probably through mediating CD8+CD69+CD103+ TRM cells, but not other renal main immune cells. These results indicated that NLRP3 inflammasome activation has a crucial role in the development and functions of CD8+CD69+CD103+ TRM cells, potentially contributing to the pathogenesis of LN. Additional research is needed to validate this thesis by generating lupus-prone mice with conditional knockout of NLRP3 in CD8+CD69+CD103+ TRM cells.

There are several potential mechanisms of NLRP3 inflammasome activation in CD8+CD69+CD103+ TRM cells in LN. One possible local signaling factor is TGF-β. TGF-β is crucial for the induction of CD103 expression and differentiation of CD103+ CD8 TRM cells in vivo, likely through downstream Smad3 activation or other indirect pathways [30]. Increased expression of TGF-β was noticed in the kidneys of MRL/lpr mice [39]. As noted in other studies [34, 40], NF-κB signaling involves NLRP3 transcription activation by TGF-β, leading to formation and activation of the NLRP3 inflammasome via Smad3-mediated ROS induction. Additionally, the role of TNFα in this process should also be considered. TNFα can interact with receptor-interacting kinase 3 and then trigger both necroptosis and NLRP3 inflammasome signaling [41, 42]. Interestingly, TNFα also helps to promote the development of TRM cells by downregulating KLF2 [43]. In addition, perforin released by CD8+ T cells has been found to trigger NLRP3 inflammasome activation in antigen-presenting cells [44]. It is important to recognize that other mechanisms might also be involved. Further genetic experiments focusing on TGF-β, TNFα, or perforin deletion in CD8+ T cells are necessary to elucidate these mechanisms.

IL-1β and IL-18 are two powerful downstream cytokines of the NLRP3 inflammasome, and they are mainly produced by innate immune cells [45]. Research has illustrated that IL-1β directly affects CD8+ T cells, improving the efficacy of their effector and memory functions [46]. Currently, intrinsic NLRP3 inflammasome activity in T cells is attracting increasing attention [13,14,15,16,17,18,19,20]. However, the inherent role of NLRP3–IL-1β signaling in CD8+ T cells is unclear. In this study, we identified the presence of enhanced autocrine NLRP3–IL-1β activity in CD8+CD69+CD103+ TRM cells. Inhibition of NLRP3 dramatically suppressed CD8+CD69+CD103+ TRM cell differentiation, and the decreased percentages of CD8+CD103+ TRM-like cells were partially reversed by the addition of IL-1β and IL-18. Our study found that the addition of IL-1β to TGF-β, but not the addition of IL-18, further promoted the differentiation of CD8+ cells into CD8+CD103+ TRM-like cells. Mechanistically, IL-1β was demonstrated to induce upregulation of TGF-βRII in CD8+ T cells via NF-κB signaling, which may account for its role in promoting TGF-β-induced TRM differentiation. This evidence suggests that autocrine NLRP3 activity in CD8+CD69+CD103+ TRM cells plays a role in establishing optimal conditions for TRM cell development and augmenting their pathogenicity. Downregulation of transcription factor KLF2 and its target gene S1PR1 plays key roles in the retention of CD8+ TRM cells [43]. IL-1β and IL-18 signaling could downregulate KLF2 expression [43, 47], which might further promote CD8+ TRM cell residency. Moreover, NLRP3 was proven to activate Smad3 downstream of TGF-β signaling and augment TGF-β signaling [48, 49]. In addition, NLRP3 can promote endogenous TGF-β expression, probably in an IL-1β–dependent manner [33, 34], which was also confirmed in our present study. The enhanced NLRP3 inflammasome activation and autocrine TGF-β production in CD8+ TRM cells might create a positive feedback loop, aiding in the maintenance of a TRM cell pool in LN. Interestingly, NLRP3 inhibition dramatically decreased active caspase-1 expression in renal CD8+CD69+CD103+ TRM cells, but it had no impact on that in CD8+CD69+CD103− TRM cells, suggesting that the NLRP3 inflammasome is specifically involved in the underlying mechanisms of CD8+CD69+CD103+ TRM cell development and activity. More evidence is required to elucidate the significance of intrinsic NLRP3 activity in CD8+CD69+CD103+ TRM cells.

T cell infiltration is an important characteristic of renal chronic inflammation in LN [50]. Patients with LN and complete clinical renal remission often exhibit persistent chronic LN activities, which are associated with a high rate of flares [51, 52]. Our previous study illustrated that long-lived and hyperactive CD8+CD103+ TRM cells actively participate in the initiation and development of LN [9]. Thus, the removal of renal T cells, especially CD8+CD103+ TRM cells, is crucial for inducing disease remission and preventing flares. In our present study, targeting the NLRP3 inflammasome markedly suppressed CD8+CD69+CD103+ TRM cell development, maintenance, and functions as well as renal damage. Collectively, these evidence indicated that NLRP3 inflammasome activation within CD8+CD69+CD103+ TRM cells is an active participant in the pathogenesis of LN. Targeting NLRP3–IL-1β signaling could alleviate disease and prevent relapses in LN by suppressing CD8+CD69+CD103+ TRM cell development and activity.

Conclusions

Our study demonstrated that NLRP3 inflammasome signaling is activated in renal CD8+CD69+CD103+ TRM cells from lupus-prone mice and human CD8+CD103+ TRM-like cells induced in vitro. Activation of the NLRP3 inflammasome in renal CD8+CD69+CD103+ TRM cells contribute to LN pathogenesis by modulating cell development and activity. Therefore, targeting NLRP3–IL-1 signaling could effectively improve renal injury and prevent flares in LN by decreasing CD8+CD69+CD103+ TRM cell infiltration and activity.

Data availability

The data used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

TRM :

Tissue-resident memory T

LN:

Lupus nephritis

GSDMD:

Gasdermin D

SLE:

Systemic lupus erythematosus

Th1:

T helper 1

Treg:

Regulatory T

Tfh:

T follicular helper

HCs:

healthy controls

PBMCs:

Peripheral blood mononuclear cells

qPCR:

Quantitative real-time PCR

ELISA:

Enzyme-linked immunosorbent assay

GZMB:

Granzyme B

BUN:

Blood urea nitrogen

PAS:

Periodic acid-Schiff

DNT:

Double-negative T

MFI:

Mean fluorescence intensity

References

  1. Siegel CH, Sammaritano LR. Systemic lupus erythematosus: a review. JAMA. 2024;331(17):1480–91.

    Article  PubMed  Google Scholar 

  2. Anders HJ, Saxena R, Zhao MH, Parodis I, Salmon JE, Mohan C. Lupus nephritis. Nat Rev Dis Primers. 2020;6(1):7.

    Article  PubMed  Google Scholar 

  3. Yu C, Li P, Dang X, Zhang X, Mao Y, Chen X. Lupus nephritis: new progress in diagnosis and treatment. J Autoimmun. 2022;132:102871.

    Article  PubMed  Google Scholar 

  4. Buggert M, Price DA, Mackay LK, Betts MR. Human circulating and tissue-resident memory CD8(+) T cells. Nat Immunol. 2023;24(7):1076–86.

    Article  PubMed  Google Scholar 

  5. Liu G, Wang Z, Li S. Heterogeneity and plasticity of tissue-resident memory T cells in skin diseases and homeostasis: a review. Front Immunol. 2024;15:1378359.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Cheuk S, Wikén M, Blomqvist L, Nylén S, Talme T, Ståhle M, et al. Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis. J Immunol. 2014;192(7):3111–20.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Chang MH, Levescot A, Nelson-Maney N, Blaustein RB, Winden KD, Morris A, et al. Arthritis flares mediated by tissue-resident memory T cells in the joint. Cell Rep. 2021;37(4):109902.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Richmond JM, Strassner JP, Rashighi M, Agarwal P, Garg M, Essien KI, et al. Resident Memory and Recirculating Memory T Cells Cooperate to maintain Disease in a mouse model of Vitiligo. J Invest Dermatol. 2019;139(4):769–78.

    Article  PubMed  Google Scholar 

  9. Zhou M, Guo C, Li X, Huang Y, Li M, Zhang T, et al. JAK/STAT signaling controls the fate of CD8(+)CD103(+) tissue-resident memory T cell in lupus nephritis. J Autoimmun. 2020;109:102424.

    Article  PubMed  Google Scholar 

  10. Liang CL, Wei YY, Chen Y, Luo Y, Qin F, Chen Y, et al. Zhen-Wu-Tang ameliorates lupus nephritis by diminishing renal tissue-resident memory CD8(+) T cells via suppressing IL-15/STAT3 pathway. Biomed Pharmacother. 2024;174:116597.

    Article  PubMed  Google Scholar 

  11. Fu J, Wu H. Structural mechanisms of NLRP3 Inflammasome Assembly and Activation. Annu Rev Immunol. 2023;41:301–16.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Huang Y, Xu W, Zhou R. NLRP3 inflammasome activation and cell death. Cell Mol Immunol. 2021;18(9):2114–27.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Arbore G, West EE, Spolski R, Robertson AAB, Klos A, Rheinheimer C, et al. T helper 1 immunity requires complement-driven NLRP3 inflammasome activity in CD4⁺ T cells. Sci (New York NY). 2016;352(6292):aad1210.

    Article  Google Scholar 

  14. Zhao Z, Xu B, Wang S, Zhou M, Huang Y, Guo C, et al. Tfh cells with NLRP3 inflammasome activation are essential for high-affinity antibody generation, germinal centre formation and autoimmunity. Ann Rheum Dis. 2022;81(7):1006–12.

    Article  PubMed  Google Scholar 

  15. Arbore G, West EE, Rahman J, Le Friec G, Niyonzima N, Pirooznia M, et al. Complement receptor CD46 co-stimulates optimal human CD8(+) T cell effector function via fatty acid metabolism. Nat Commun. 2018;9(1):4186.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Zhou M, Li M, Guo C, Wang S, Li X, Zhao S, et al. Circulating TFH cells is correlated with disease activity in anti-MDA5 antibody positive idiopathic inflammatory myopathies. Clin Exp Rheumatol. 2021;39(4):804–10.

    Article  PubMed  Google Scholar 

  17. Bruchard M, Rebé C, Derangère V, Togbé D, Ryffel B, Boidot R, et al. The receptor NLRP3 is a transcriptional regulator of TH2 differentiation. Nat Immunol. 2015;16(8):859–70.

    Article  PubMed  Google Scholar 

  18. Park SH, Ham S, Lee A, Möller A, Kim TS. NLRP3 negatively regulates Treg differentiation through Kpna2-mediated nuclear translocation. J Biol Chem. 2019;294(47):17951–61.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Martin BN, Wang C, Zhang CJ, Kang Z, Gulen MF, Zepp JA, et al. T cell-intrinsic ASC critically promotes T(H)17-mediated experimental autoimmune encephalomyelitis. Nat Immunol. 2016;17(5):583–92.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Zhao C, Gu Y, Zeng X, Wang J. NLRP3 inflammasome regulates Th17 differentiation in rheumatoid arthritis. Clin Immunol (Orlando Fla). 2018;197:154–60.

    Article  Google Scholar 

  21. Oliveira CB, Lima CAD, Vajgel G, Sandrin-Garcia P. The role of NLRP3 inflammasome in Lupus Nephritis. Int J Mol Sci. 2021;22(22).

  22. Fu R, Guo C, Wang S, Huang Y, Jin O, Hu H, et al. Podocyte activation of NLRP3 inflammasomes contributes to the development of Proteinuria in Lupus Nephritis. Arthritis Rheumatol. 2017;69(8):1636–46.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Guo C, Fu R, Zhou M, Wang S, Huang Y, Hu H, et al. Pathogenesis of lupus nephritis: RIP3 dependent necroptosis and NLRP3 inflammasome activation. J Autoimmun. 2019;103:102286.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Lech M, Lorenz G, Kulkarni OP, Grosser MO, Stigrot N, Darisipudi MN, et al. NLRP3 and ASC suppress lupus-like autoimmunity by driving the immunosuppressive effects of TGF-β receptor signalling. Ann Rheum Dis. 2015;74(12):2224–35.

    Article  PubMed  Google Scholar 

  25. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40(9):1725.

    Article  PubMed  Google Scholar 

  26. Gladman DD, Ibañez D, Urowitz MB. Systemic lupus erythematosus disease activity index 2000. J Rheumatol. 2002;29(2):288–91.

    PubMed  Google Scholar 

  27. Ye B, Chen B, Guo C, Xiong N, Huang Y, Li M, et al. C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis. Mol Ther. 2024;32(5):1540–60.

    Article  PubMed  Google Scholar 

  28. Zeng Q, Wang S, Li M, Wang S, Guo C, Ruan X, et al. Spleen fibroblastic reticular cell-derived acetylcholine promotes lipid metabolism to drive autoreactive B cell responses. Cell Metab. 2023;35(5):837–e548.

    Article  PubMed  Google Scholar 

  29. Bergsbaken T, Bevan MJ, Fink PJ. Local inflammatory cues regulate differentiation and persistence of CD8(+) tissue-Resident memory T cells. Cell Rep. 2017;19(1):114–24.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Qiu Z, Chu TH, Sheridan BS. TGF-β: many paths to CD103(+) CD8 T cell residency. Cells. 2021;10(5).

  31. Tieu R, Zeng Q, Zhao D, Zhang G, Feizi N, Manandhar P, et al. Tissue-resident memory T cell maintenance during antigen persistence requires both cognate antigen and interleukin-15. Sci Immunol. 2023;8(82):eadd8454.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Hirai T, Yang Y, Zenke Y, Li H, Chaudhri VK, De La Cruz Diaz JS, et al. Competition for active TGFβ cytokine allows for Selective Retention of Antigen-Specific tissue- Resident memory T cells in the epidermal niche. Immunity. 2021;54(1):84–e985.

    Article  PubMed  Google Scholar 

  33. Xu J, Chen P, Luan X, Yuan X, Wei S, Li Y, et al. The NLRP3 activation in infiltrating macrophages contributes to corneal fibrosis by inducing TGF-β1 expression in the corneal epithelium. Invest Ophthalmol Vis Sci. 2022;63(8):15.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Kang H, Seo E, Oh YS, Jun HS. TGF-β activates NLRP3 inflammasome by an autocrine production of TGF-β in LX-2 human hepatic stellate cells. Mol Cell Biochem. 2022;477(5):1329–38.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Rex J, Lutz A, Faletti LE, Albrecht U, Thomas M, Bode JG, et al. IL-1β and TNFα differentially influence NF-κB activity and FasL-Induced apoptosis in primary murine hepatocytes during LPS-Induced inflammation. Front Physiol. 2019;10:117.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Chen Y, Yan Y, Liu H, Qiu F, Liang CL, Zhang Q, et al. Dihydroartemisinin ameliorates psoriatic skin inflammation and its relapse by diminishing CD8(+) T-cell memory in wild-type and humanized mice. Theranostics. 2020;10(23):10466–82.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Li L, Tang W, Zhang Y, Jia M, Wang L, Li Q, et al. Targeting tissue-resident memory CD8(+) T cells in the kidney is a potential therapeutic strategy to ameliorate podocyte injury and glomerulosclerosis. Mol Ther. 2022;30(8):2746–59.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Christo SN, Park SL, Mueller SN, Mackay LK. The multifaceted role of tissue-Resident memory T cells. Annu Rev Immunol. 2024;42(1):317–45.

    Article  PubMed  Google Scholar 

  39. Huang C, Meng M, Li S, Liu S, Li L, Su Y, et al. Umbilical cord mesenchymal stem cells ameliorate kidney Injury in MRL/Ipr mice through the TGF-β1 pathway. Front Cell Dev Biol. 2022;10:876054.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Sun J, Ge X, Wang Y, Niu L, Tang L, Pan S. USF2 knockdown downregulates THBS1 to inhibit the TGF-β signaling pathway and reduce pyroptosis in sepsis-induced acute kidney injury. Pharmacol Res. 2022;176:105962.

    Article  PubMed  Google Scholar 

  41. He S, Wang L, Miao L, Wang T, Du F, Zhao L, et al. Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha. Cell. 2009;137(6):1100–11.

    Article  PubMed  Google Scholar 

  42. Lawlor KE, Khan N, Mildenhall A, Gerlic M, Croker BA, D’Cruz AA, et al. RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL. Nat Commun. 2015;6:6282.

    Article  PubMed  Google Scholar 

  43. Skon CN, Lee JY, Anderson KG, Masopust D, Hogquist KA, Jameson SC. Transcriptional downregulation of S1pr1 is required for the establishment of resident memory CD8 + T cells. Nat Immunol. 2013;14(12):1285–93.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Yao Y, Chen S, Cao M, Fan X, Yang T, Huang Y, et al. Antigen-specific CD8(+) T cell feedback activates NLRP3 inflammasome in antigen-presenting cells through perforin. Nat Commun. 2017;8:15402.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Rao DA, Tracey KJ, Pober JS. IL-1alpha and IL-1beta are endogenous mediators linking cell injury to the adaptive alloimmune response. J Immunol. 2007;179(10):6536–46.

    Article  PubMed  Google Scholar 

  46. Sarkar S, Yuzefpolskiy Y, Xiao H, Baumann FM, Yim S, Lee DJ, et al. Programming of CD8 T cell quantity and polyfunctionality by direct IL-1 signals. J Immunol. 2018;201(12):3641–50.

    Article  PubMed  Google Scholar 

  47. Liang Y, Luo J, Yang N, Wang S, Ye M, Pan G. Activation of the IL-1β/KLF2/HSPH1 pathway promotes STAT3 phosphorylation in alveolar macrophages during LPS-induced acute lung injury. Biosci Rep. 2020;40(3).

  48. Wu M, Han W, Song S, Du Y, Liu C, Chen N, et al. NLRP3 deficiency ameliorates renal inflammation and fibrosis in diabetic mice. Mol Cell Endocrinol. 2018;478:115–25.

    Article  PubMed  Google Scholar 

  49. Song S, Qiu D, Luo F, Wei J, Wu M, Wu H, et al. Knockdown of NLRP3 alleviates high glucose or TGFB1-induced EMT in human renal tubular cells. J Mol Endocrinol. 2018;61(3):101–13.

    Article  PubMed  Google Scholar 

  50. Lech M, Anders H-J. The pathogenesis of lupus nephritis. J Am Soc Nephrol. 2013;24(9):1357–66.

    Article  PubMed  PubMed Central  Google Scholar 

  51. De Rosa M, Azzato F, Toblli JE, De Rosa G, Fuentes F, Nagaraja HN, et al. A prospective observational cohort study highlights kidney biopsy findings of lupus nephritis patients in remission who flare following withdrawal of maintenance therapy. Kidney Int. 2018;94(4):788–94.

    Article  PubMed  Google Scholar 

  52. Winchester R, Wiesendanger M, Zhang H-Z, Steshenko V, Peterson K, Geraldino-Pardilla L, et al. Immunologic characteristics of intrarenal T cells: trafficking of expanded CD8 + T cell β-chain clonotypes in progressive lupus nephritis. Arthritis Rheum. 2012;64(5):1589–600.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We sincerely appreciate all participants in the study. The graphical abstract and Figs. 1A and 2A and F and S2A were created with BioRender.com, and we acknowledge its valuable contribution to our work. The specific citations for these figures are as follows: Graphical abstract, Created in BioRender. Zhang, H. (2024) https://BioRender.com/m61q624; Fig. 1A, Created in BioRender. Zhang, H. (2024) https://BioRender.com/r09m405; Fig. 2A, Created in BioRender. Zhang, H. (2024) https://BioRender.com/i40w123; Fig. 2F, Created in BioRender. Zhang, H. (2024) https://BioRender.com/g26v870; Figure S2A, Created in BioRender. Zhang, H. (2024) https://BioRender.com/g61i184.

Funding

This study was funded by the National Natural Science Foundation of China (82101884, 82171770, 81971519) and the Chinese Postdoctoral Science Foundation (2021M693661).

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Author notes

  1. Yimei Lai, Lili Zhuang and Jieying Zhu contributed equally to this work.

Authors and Affiliations

  1. Department of Rheumatology, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Second Road, Guangzhou, 510080, P. R. China

    Yimei Lai, Lili Zhuang, Jieying Zhu, Shuang Wang, Chaohuan Guo, Binfeng Chen, Jin Li, Jia Shi, Mengyuan Li, Niansheng Yang & Mianjing Zhou

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  1. Yimei Lai
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Contributions

MZ, NY, YL, LZ, and JZ designed the whole study. CG and BC performed the laboratory work. SW, ML, LZ, and JZ did the statistical analyses and data interpretation. JS and ML collected clinical samples and clinical information. NY and MZ wrote the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Niansheng Yang or Mianjing Zhou.

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This study was approved by the ethical review board of The First Affiliated Hospital, Sun Yat-sen University. All participants provided written informed consent.

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12967_2024_5951_MOESM1_ESM.png

Additional file 1: Graphical abstract: NLRP3 inflammasome pathways are activated in renal CD8+CD69+CD103+ TRM cells in lupus-prone mice

NLRP3 inflammasome regulates CD8+CD103+ TRM cell differentiation and effector functions

Inhibition of NLRP3 inflammasome suppresses CD8+ TRM cell response and alleviates renal disease of LN

IL-1β promotes the expression of TGF-βRII in CD8+ T cells via NF-ĸB signaling.

12967_2024_5951_MOESM2_ESM.png

Additional file 2: Figure S1: Enhanced CD49a and CXCR3 expression in renal CD8+CD69+CD103+ TRM cells of lupus-prone mice. The kidneys of 18-week-old MRL/lpr mice were harvested to prepare single-cell suspensions. The expression of CD49a (A and B) and CXCR3 (C and D) was assessed by flow cytometry. Representative plots focusing on CD8+CD69−CD103−, CD8+CD69+CD103− and CD8+CD69+CD103+ cells are presented. Data are presented as mean ± SEM from five mice. *p < 0.05, ***p < 0.001, one-way ANOVA corrected for multiple comparisons. ns, not significant.

12967_2024_5951_MOESM3_ESM.png

Additional file 3: Figure S2: TGF-β alone or in combination with IL-15 induced active CD8+CD103+ TRM−like cells. CD8+ T cells isolated from the PBMCs of HCs were triggered by anti-CD3/CD28 Dynabeads and 10 ng/ml TGF-β alone or in combination with 10 ng/ml IL-15 for 3 days. A: Experiment scheme. B and C: CD103 expression on CD8+ T cells was assessed by flow cytometry. Representative contour plots and percentages of CD8+CD103+ T cells are displayed. D–G: Annexin V, 7AAD (D and E) and Ki-67 (F and G) expression on CD8+CD103+ T cells was assessed by flow cytometry. Representative contour plots and percentages are displayed. H and I: CD49a, CXCR3, S1PR1 and CD62L expression was analyzed by flow cytometry, concentrating on CD8+CD103− and CD8+CD103+ cells. All data are presented as the mean ± SEM of five independent assays. ***p < 0.001, and ****p < 0.0001, one-way ANOVA corrected for multiple comparisons (C–G), two-way ANOVA corrected for multiple comparisons (I). ns, not significant.

12967_2024_5951_MOESM4_ESM.png

Additional file 4: Figure S3: Coomassie Brilliant Blue staining of proteins in CD8+CD103− and CD8+CD103+ T Cells. CD8+ T cells, isolated from the PBMCs of patients with LN or HCs, were triggered with anti-CD3/CD28 Dynabeads in the presence of 10 ng/ml TGF-β for 3 days and then sorted into CD8+CD103− and CD8+CD103+ populations. Proteins extracted from lysed cells were subjected to electrophoresis, and the resulting gel was stained with Coomassie Brilliant Blue. A: A representative Coomassie Brilliant Blue staining of CD8+CD103− and CD8+CD103+ cells derived from HCs is shown. B: A representative Coomassie Brilliant Blue staining of CD8+CD103+ cells derived from patients with LN or HCs is shown.

12967_2024_5951_MOESM5_ESM.png

Additional file 5: Figure S4: Human CD8+CD103+ TRM-like cells induced by TGF-β demonstrated enhanced proliferative capabilities and functionality. CD8+ T cells isolated from the PBMCs of HCs were incubated with anti-CD3/CD28 Dynabeads and 10 ng/ml TGF-β for 3 days to induce the differentiation of CD103+ cells. A–I: Ki-67 (A and B), perforin (C and D), GZMB (E and F), TNFα and IFNγ (G–I) expression was assessed by flow cytometry, concentrating on CD8+CD103− and CD8+CD103+ cells. All data are displayed as the mean ± SEM of five independent samples. *p < 0.05, **p < 0.01, and ***p < 0.001, paired Student’s t-test.

12967_2024_5951_MOESM6_ESM.png

Additional file 6: Figure S5: Gating strategy. Single-cell suspensions were isolated from mouse kidneys. Flow cytometry was used to analyze the cells. Dead cells were initially excluded by zombie staining. CD45+ cells were selected and further sorted into CD3+CD8+, CD3+CD4+, and CD3+CD4−CD8− DNT cell populations for further analysis. Among CD45+ cells, CD19+ cells were identified as B cells, and CD68+ cells were selected as macrophages.

12967_2024_5951_MOESM7_ESM.png

Additional file 7: Figure S6: The impact of MCC950 on NLRP3 pathway in renal main immune cells of MRL/lpr mice. MRL/lpr mice were treated with MCC950 (10 mg/kg) or vehicle for 6 weeks starting at 12 weeks of age. Kidneys were collected to prepare single-cell suspensions. Cells were sorted according to CD45+ expression for further study. Flow cytometry was utilized to evaluate active caspase-1 levels in CD3+CD8+ T cells (A and B), CD3+CD4+ T cells (C and D), CD3+CD4−CD8− DNT cells (E and F), CD19+ B cells (G and H), and CD68+ macrophages (I and J). All data are displayed as the mean ± SEM. Data were collected from eight mice. *p < 0.05, **p < 0.01, unpaired Student’s t-test. ns, not significant.

12967_2024_5951_MOESM8_ESM.png

Additional file 8: Figure S7: The effects of different concentrations of MCC950 or Ac-YVAD-cmk on the differentiation of CD8+CD103+ cells. CD8+ T cells isolated from the PBMCs of HCs were treated with anti-CD3/CD28 Dynabeads and 10 ng/ml TGF-β for 3 days to induce CD103+ cell differentiation. Cells were treated with different concentrations of MCC950 (A and B) or Ac-YVAD-cmk (C and D) as indicated. CD103 expression was analyzed by flow cytometry, concentrating on CD8+ cells. Representative plots were presented. All data are displayed as the mean ± SEM. Data were collected from five independent experiments. **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA corrected for multiple comparisons.

12967_2024_5951_MOESM9_ESM.png

Additional file 9: Figure S8: MCC950 treatment mitigated the elevation of IL-1β–dependent TGF-β expression in CD8+CD103+ TRM cells. A and B: Eighteen-week-old MRL/lpr mice were sacrificed. Kidneys were collected, and single-cell suspensions were generated. TGF-β expression was evaluated by flow cytometry. Representative gated plots were analyzed for CD8+CD69−CD103−, CD8+CD69+CD103− and CD8+CD69+CD103+ cells. Data were obtained from eight mice. C and D: CD8+ T cells isolated from the PBMCs of HCs were treated with anti-CD3/CD28 Dynabeads in the absence or presence of 10 ng/ml TGF-β for 3 days. TGF-β expression was analyzed by flow cytometry, concentrating on CD8+CD103− and CD8+CD103+ cells cultured with TGF-β, or CD8+ T cells cultured without TGF-β. Data were collected from five independent experiments. E and F: MRL/lpr mice were treated with 10 mg/kg MCC950 or vehicle for 6 weeks starting at 12 weeks of age. Kidneys were obtained to prepare single-cell suspensions. TGF-β expression was determined by flow cytometry, focusing on CD8+CD69+CD103+ TRM cells. All data were collected from eight mice. G and H: CD8+ T cells isolated from the PBMCs of HCs were treated with anti-CD3/CD28 Dynabeads and 10 ng/ml TGF-β for 3 days to induce the differentiation of CD103+ cells. In certain experiments, an IL-1 receptor antagonist (20 µg/ml) was also included. TGF-β expression was quantified by flow cytometry, concentrating on CD8+CD103+ cells (n = 5). All data are displayed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA corrected for multiple comparisons (B), paired Student’s t-test (D), unpaired Student’s t-test (F–H).

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Lai, Y., Zhuang, L., Zhu, J. et al. Novel approach to alleviate lupus nephritis: targeting the NLRP3 inflammasome in CD8+CD69+CD103+ TRM cells. J Transl Med 22, 1139 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-024-05951-9

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Journal of Translational Medicine

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