Heat-killed Lactobacillus johnsonii Directly Inhibits Th17 Cell Effector Function via Selective Translational Suppression
Chin-Ning Chen, Selga I. Jansons, Faye Sun, Kimberly M. Bonger, Bethany B. Moore, Xiaofeng Zhou
TL;DR
Heat-killed Lactobacillus johnsonii suppresses IL-17A production in Th17 cells without killing them, offering a new way to control inflammation.
Contribution
HK Lj selectively inhibits IL-17A translation in Th17 cells, independent of antigen-presenting cells or receptor signaling.
Findings
HK Lj directly suppresses IL-17A production in iTh17 cells without affecting their viability.
The suppression occurs at the translational level without altering mRNA or other cytokines.
The mechanism is independent of Toll-like or C-type lectin receptor signaling.
Abstract
Interleukin-17A (IL-17A)–producing Th17 cells are essential for mucosal host defense but also drive chronic inflammation and fibrosis when dysregulated. While commensal Lactobacillus species modulate Th17 responses through antigen-presenting cells, their direct effects on differentiated Th17 cells are poorly understood. We demonstrate that heat-killed Lactobacillus johnsonii XZ17 (HK Lj) directly suppresses IL-17A production in induced Th17 (iTh17) cells, independent of antigen-presenting cells. Unlike live L. johnsonii, which reduces IL-17A via environmental acidification and cell death, HK Lj preserves iTh17 viability. Mechanistically, HK Lj selectively inhibits IL-17A translation without altering II17a or Rorc mRNA levels, global protein synthesis, or other cytokines like IL-10. This suppression is independent of Toll-like or C-type lectin receptor signaling. These findings reveal a…
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TopicsPsoriasis: Treatment and Pathogenesis · Probiotics and Fermented Foods · Pediatric health and respiratory diseases
Introduction
Mucosal surfaces are colonized by diverse communities of commensal microorganisms that play essential roles in shaping host immune responses. Among these, Lactobacillus species are prominent members of the gastrointestinal and respiratory microbiota and have been widely implicated in the maintenance of immune homeostasis and protection against inflammatory disease^1–4^. Through interactions with host immune cells, Lactobacillus spp. can modulate cytokine production, barrier function, and antimicrobial defenses at mucosal sites^5–7^.
Th17 cells represent a critical link between commensal sensing and mucosal immunity. Characterized by their production of interleukin-17A (IL-17A), Th17 cells contribute to host defense against extracellular pathogens but can also drive chronic inflammation, fibrosis, and autoimmunity when dysregulated^8–10^. The microbiota is a major determinant of Th17 cell differentiation and function, and several Lactobacillus species have been shown to modulate Th17 responses in vivo^3,11,12^. However, these effects are generally attributed to indirect mechanisms mediated by antigen-presenting cells, particularly dendritic cells and macrophages.
Lactobacillus johnsonii has recently emerged as a commensal with potent immunomodulatory properties^13^. Prior studies indicate that L. johnsonii regulates inflammatory responses through interactions with innate immune cells, leading to altered T cell polarization and cytokine production^13,14^. We recently isolated L. johnsonii strain XZ17 from the lungs of healthy mice and demonstrated that intranasal administration of heat-killed L. johnsonii XZ17 (HK Lj) attenuates virus-induced pulmonary fibrosis in bone marrow transplanted (BMT) mice by suppressing IL-17A production in vivo, in part through PD-L1/PD-1 signaling^15,16^. Whether HK Lj can directly modulate Th17 cells, independent of antigen-presenting cells, remains unknown.
In the present study, we investigate the direct effects of L. johnsonii XZ17 on Th17 cells using a reductionist in vitro system. We show that, unlike live bacteria, HK Lj suppresses IL-17A production without compromising Th17 cell viability. Mechanistically, HK Lj selectively inhibits IL-17A translation without altering II17a transcription or global protein synthesis. These findings reveal a previously unrecognized, T cell–intrinsic mechanism by which a commensal-derived signal fine-tunes Th17 effector function.
Results
Live L. johnsonii XZ17 suppresses IL-17A production by iTh17 cells through environmental acidification
To investigate whether live L. johnsonii XZ17 modulates the activity of Th17 cells, we cocultured induced Th17 (iTh17) cells with varying concentrations of live L. johnsonii XZ17 overnight and measured IL-17A secretion in the medium using ELISA. iTh17 cells were differentiated from naïve CD4^+^ T cells isolated from the spleens of C57BL/6J mice by stimulation with pro-Th17 cytokines according to established protocols^17^. Coculture with L. johnsonii XZ17 resulted in a significant, dose-dependent reduction in IL-17A production by iTh17 cells (Fig. 1A).
Contrary to the common presumption that Lactobacillus spp. are generally non-toxic to immune cells^5,18^, increasing concentrations of L. johnsonii XZ17 negatively impacted iTh17 cell viability (Fig. 1B). Since Lactobacillus spp. rapidly produces lactic acid and can decrease the pH of well-buffered culture media^19,20^, we hypothesized that environmental acidification may mediate the observed effects. To test this, we cultured iTh17 cells in a 1:1 mixture of L. johnsonii XZ17-conditioned media and complete media (pH ≈ 5.5), which resulted in a marked reduction in IL-17A production. (Fig. 1C) and decreased cell viability (Fig. 1D). Importantly, neutralizing the pH of conditioned medium to 7 with NaOH improved both IL-17A production and cell viability to levels comparable to untreated controls (Fig. 1C, D). Collectively, these results demonstrate that live L. johnsonii XZ17 suppresses IL-17A production by iTh17 cells primarily by acidifying the environment, which in turn reduces cell viability and cytokine secretion.
Heat-killed L. johnsonii XZ17 suppresses IL-17A production without reducing iTh17 cell viability
Since live L. johnsonii XZ17 appears to suppress IL-17A production primarily through environmental acidification and associated reductions in cell viability, we sought to determine whether heat-killed L. johnsonii XZ17 (HK Lj) could modulate IL-17A expression in iTh17 cells without compromising their survival. Notably, we previously observed that intranasal administration of HK Lj suppressed pulmonary IL-17A levels in BMT mice infected with murine gammaherpesvirus 68^16^.
To assess the direct effects of HK Lj on iTh17 cells, we cocultured iTh17 cells with HK Lj at varying ratios. Remarkably, HK Lj significantly suppressed IL-17A production even at a relatively low cell-to-bacteria ratio of 1:5 (Fig. 2A), while iTh17 cell viability remained unchanged (Fig. 2B). These data suggest that HK Lj can downregulate IL-17A production by iTh17 cells without negatively affecting their viability, indicating a cell-intrinsic immunomodulatory effect that is independent of environmental acidification.
IL-17A production is not regulated by HK Lj at the transcriptional level
Cytokine production is tightly regulated, typically at the transcriptional level, to ensure an appropriate immune response^21^. Unexpectedly, while protein levels of IL-17A in the culture medium of iTh17 cells were significantly reduced following overnight treatment with HK Lj (Fig. 3A), the mRNA levels of II17a in these cells did not show a significant change (Fig. 3B). RORγt, a master transcription factor encoded by Rorc gene governing Th17 cell differentiation and II17a expression^9^, also exhibited similar mRNA levels in both treated and untreated iTh17 cells (Fig. 3C). These observations indicate that HK Lj suppresses IL-17A production in iTh17 cells via mechanisms independent of transcriptional regulation of II17a or Rorc.
HK Lj regulates IL-17A production at the translational level
In addition to transcriptional regulation, cytokine production can be influenced at the level of protein translation or secretion. To determine whether HK Lj modulates IL-17A production through these mechanisms, we performed intracellular staining using fluorescent anti-IL-17A antibodies and analyzed iTh17 cells by fluorescence microscopy or flow cytometry.
If HK Lj blocked IL-17A secretion, intracellular accumulation of the cytokine would be expected to lead to a higher fluorescence signal. Conversely, if translation were inhibited, a reduction in intracellular IL-17A fluorescence would be observed. Microscopy demonstrated that HK Lj-treated iTh17 cells did not show accumulation of intracellular IL-17A, rather they exhibited slightly lower intracellular IL-17A staining compared to untreated controls, suggesting inhibition at the translational level rather than at secretion (Fig. 4A). Notably, iTh17 cells with larger nuclei (DAPI staining) often showed reduced intracellular IL-17A fluorescence (Fig. 4A). To further investigate the role of secretion, we treated cells with GolgiStop to inhibit protein export. Under these conditions, the difference in IL-17A accumulation between HK Lj-treated and untreated iTh17 cells became more pronounced (Fig. 4B).
Flow cytometry provided additional confirmation. Gated iTh17 cells (see Supplementary Fig. 1) were distinguished as IL-17A^low^ large cells (population a) and IL-17A^+^ small cells (population b) (Fig. 4C). Consistent with microscopy, HK Lj treatment significantly decreased IL-17A staining intensity in both populations, as demonstrated by histogram plots (Fig. 4D) and mean fluorescence intensity (MFI) measurements (Fig. 4E). Collectively, these findings strongly indicate that HK Lj suppresses IL-17A production by iTh17 cells predominantly through inhibition at the translational level.
HK Lj selectively regulates IL-17A translation without global suppression of protein synthesis
To determine whether HK Lj’s regulation of protein translation in iTh17 cells is selective for IL-17A or affects global protein synthesis, we employed noncanonical amino acid tagging to label newly synthesized proteins^22^. β-ethynylserine (βES), a threonine analog containing an alkyne group, was incorporated into newly synthesized proteins during a 30-minute incubation. A fluorescent azide was then covalently attached to βES, enabling flow cytometric analysis of nascent protein production (Fig. 5A). Cycloheximide (CHX), a well-established eukaryotic translation inhibitor, served as the negative control.
Among gated iTh17 cells, the IL-17A^+^ population actively synthesized new proteins, whereas the IL-17A^low^ cells exhibited minimal protein synthesis during the 30-minute incubation period (Fig. 5A). HK Lj treatment resulted in a decrease in IL-17A staining intensity in IL-17A^+^ cells, as evidenced by both histogram plots (Fig. 5B, left) and MFI (Fig. 5C, left). In contrast, no significant difference was observed in the levels of total newly synthesized protein between HK Lj-treated and untreated iTh17 cells (Fig. 5B, right; Fig. 5C, right). To further verify the specificity of HK Lj’s translational regulation, we measured production of IL-10, a cytokine unrelated to the IL-17 family, by ELISA. IL-10 concentrations in the culture media were comparable between HK Lj-treated and untreated iTh17 cells (Supplementary Fig. 2). Taken together, these results indicate that HK Lj specifically suppresses IL-17A translation in iTh17 cells, without affecting global protein synthesis or the production of other cytokines such as IL-10.
HK Lj’s effects on iTh17 cells are optimized by, but do not require TLR or CLR signaling
Previous studies, including our own, have demonstrated that Lactobacilli modulate cytokine production in CD4^+^ T cells primarily via dendritic cells and other antigen-presenting cells^5,16^. However, how Lactobacillus or their components directly regulate cytokine production by CD4^+^ T cells remains unclear. Since CD4^+^ T cells express both Toll-like receptors (TLRs) and C-type lectin receptors (CLRs)^23,24^, we investigated whether HK Lj’s regulation of IL-17A production depends on these pathways.
To assess TLR involvement, we differentiated iTh17 cells from Myd88 knockout mice, as MYD88 is the key adaptor for signaling through most TLRs except TLR3^25^. Myd88-deficient iTh17 cells displayed reduced baseline IL-17A production compared to wild-type, but their IL-17A levels were still significantly decreased upon HK Lj treatment (Fig. 6A). For CLR signaling, we inhibited spleen tyrosine kinase (Syk)—a central downstream mediator of CLR signaling—using the Syk inhibitor R406^26,27^. While Syk inhibition similarly reduced baseline IL-17A production, it did not prevent further IL-17A suppression by HK Lj (Fig. 6B). Collectively, these results indicate that TLR and CLR signaling pathways enhance, but are not essential for, the HK Lj-mediated downregulation of IL-17A production in iTh17 cells.
Discussion
In this study, we identify a previously unrecognized mechanism by which a commensal-derived stimulus directly modulates Th17 effector function. We show that heat-killed Lactobacillus johnsonii XZ17 suppresses IL-17A production in induced Th17 cells through selective inhibition of cytokine translation, without affecting cell viability, lineage-associated transcription, or global protein synthesis. Importantly, this effect occurs in the absence of dendritic cells or other antigen-presenting cells, demonstrating a T cell–intrinsic mode of regulation.
Most studies examining Lactobacillus-mediated immune regulation have focused on indirect mechanisms involving antigen-presenting cells, particularly dendritic cells and macrophages, which shape CD4^+^ T cell differentiation and cytokine profiles^5,6,28^. In contrast, our data demonstrate that HK Lj acts directly on differentiated Th17 cells. This distinction is notable, as Th17 cells reside at mucosal surfaces where they may encounter bacterial components independent of classical antigen presentation^1,2,29^.
Our findings also contrast with the effects of live L. johnsonii, which suppresses IL-17A production primarily through environmental acidification that compromises T cell viability. Heat killing eliminates this confounding variable, revealing a biologically active signal capable of modulating Th17 effector function without inducing cellular stress or death. These observations suggest that structural components of L. johnsonii, rather than metabolic byproducts, are sufficient to directly regulate Th17 cells.
A central conceptual advance of this study is the identification of translational control as a mechanism by which a commensal-derived signal selectively restrains IL-17A production. While transcriptional regulation of II17a by RORγt and associated signaling pathways is well established^9,10^, post-transcriptional regulation of IL-17A in Th17 cells has received comparatively limited attention. We show that HK Lj markedly reduces intracellular and secreted IL-17A protein without altering II17a or Rorc mRNA levels, strongly supporting translational inhibition.
Notably, HK Lj does not suppress global protein synthesis or the production of an unrelated cytokine, IL-10, indicating that this effect is highly selective rather than a consequence of generalized translational arrest. Such selectivity suggests that IL-17A translation may be particularly sensitive to modulation by microbial signals. These findings add an additional layer to the current paradigm of Th17 regulation and highlight translation as a tunable checkpoint for inflammatory cytokine output.
The selective nature of HK Lj–mediated IL-17A suppression suggests several avenues for future investigation. Defining the molecular machinery responsible for IL-17A translational repression, such as RNA-binding proteins^30^, microRNAs^31^, or signaling pathways regulating ribosome engagement^32^, will be essential for understanding how Th17 effector functions are fine-tuned. In parallel, biochemical fractionation of HK Lj may identify the bacterial components responsible for this effect.
CD4^+^ T cells express multiple pattern-recognition receptors, including Toll-like receptors and C-type lectin receptors, which can influence cytokine production and effector function^23,24^. Although genetic deletion of Myd88 or pharmacologic inhibition of Syk reduced baseline IL-17A production, neither intervention abrogated the suppressive effect of HK Lj. These results indicate that canonical TLR and CLR pathways enhance Th17 cytokine production but are not strictly required for HK Lj–mediated translational repression.
This finding raises the possibility that Th17 cells sense HK Lj through alternative mechanisms, such as noncanonical receptors^33^, membrane perturbation^34^, or signaling pathways linked to cellular stress responses or metabolic regulation^35^. Identifying the upstream sensing pathways will be an important goal of future work.
From a translational perspective, IL-17A is a key pathogenic driver in autoimmune, inflammatory, and fibrotic diseases, yet systemic IL-17 blockade carries significant risks^10^. The ability of HK Lj to selectively dampen IL-17A production without broadly suppressing T cell function highlights its potential as a targeted immunomodulatory strategy. Together with our previous in vivo findings demonstrating protection against virus-induced lung fibrosis^16^, this work supports the concept that heat-killed commensals can be harnessed to modulate pathological inflammation through precise, cell-intrinsic mechanisms.
Material and Methods
Induced Th17 cell culture
Naïve CD4^+^ T cells were isolated from spleens of 6–8 week old C57BL/6J mice (JAX000664, the Jackson Laboratory) using the EasySep^™^ Mouse Naïve CD4^+^ T Cell Isolation Kit (STEMCELL Technologies), following the manufacturer’s instructions. Isolated cells were seeded in 96-well plates (Fisher Brand) at a density of 2 × 10^5^ cells per well and activated with 200 μL of 2.5 μg/mL plate-bound anti-CD3 antibody (Invitrogen) and 5 μg/mL soluble anti-CD28 antibody (BioLegend) iTh17 differentiation medium. The differentiation medium consisted of Iscove’s Modified Dulbecco’s Medium (IMDM; Gibco^™^ Thermo Fisher Scientific) supplemented with 10% fetal bovine serum fetal bovine serum (FBS, Gibco^™^), 1% L-glutamine, 5 μg/mL anti-mIL-4 antibody (Invitrogen), 0.1 μg/mL recombinant mouse IL-6 (BioLegend), 0.01 μg/mL recombinant mouse TGF-β (R&D Systems), 0.02 μg/mL recombinant mouse IL-23 (BioLegend), and 10 μg/mL anti-IFN-γ antibody (BioLegend). For experiments involving live L. johnsonii XZ17, penicillin and streptomycin were omitted from the culture medium. Cells were incubated for three days to generate induced Th17 (iTh17) cells. Subsequently, the medium was replaced with fresh complete IMDM supplemented with IL-23 for further assays. Undifferentiated naïve CD4^+^ T cells were included as controls. All procedures involving mice were approved by the University of Michigan’s Institutional Animal Care and Use Committee, as per the protocol numbers PRO00010437 and PRO00011303.
Preparation of L. johnsonii XZ17
The L. johnsonii XZ17 strain was isolated from a healthy naïve adult C57BL/6J mouse^15,16^. For stock preparation, 100 mL of de Man, Rogosa, and Sharpe (MRS) broth (BD Biosciences) was inoculated with bacteria from a frozen glycerol stock and cultured statically overnight at 37°C. Stationary-phase cultures were harvested by centrifugation at 1,500 × g for 5 minutes at 4°C and resuspended in 50 mL of a 1:1 (vol/vol) solution of MRS broth and 50% glycerol. Aliquots of 500 μL were stored at −80°C. The viable cell count of prepared stocks was determined to be 1 × 10^9^ CFU/vial by serial dilution.
To prepare live L. johnsonii XZ17 for experiments, frozen stocks were rapidly thawed at 37°C, pelleted by centrifugation at 1,500 × g for 2 minutes, and washed with PBS to remove excess glycerol. The cells were resuspended in IMDM supplemented with 10% fetal bovine serum (FBS, Gibco^™^ ThermoFisher Scientific) at a final concentration of 1 × 10^6^ CFU/μL. This suspension was added to iTh17 cell cultures (without antibiotics) to achieve the designated final iTh17 cell-to-bacteria ratio. Bacterial viability was confirmed throughout the experiments by plating aliquots on MRS agar.
To generate heat-killed L. johnsonii XZ17, a vial of frozen stock was thawed at 37°C, washed with sterile PBS, resuspended in 1 mL of room temperature PBS, and incubated in a 70°C water bath for 15 minutes to ensure bacterial inactivation. The heat-killed cells were then pelleted, resuspended in sterile PBS to a final concentration equivalent to 1 × 10^6^ CFU/μL (pre-inactivation), and aliquots of 50 μL were stored at −80°C. Complete inactivation was confirmed by the absence of growth on MRS agar after two days at 37°C. Appropriate volumes of HK Lj were added to iTh17 cultures to achieve the designated final cell-to-bacteria ratio.
Culturing iTh17 cells in L. johnsonii XZ17-conditioned medium
An overnight culture of L. johnsonii XZ17 in MRS broth was pelleted and washed with PBS as described above. The bacteria were then resuspended in IMDM supplemented with 10% FBS at a concentration of 1 × 10^7^ CFU/mL (equivalent to 10 times the number of iTh17 cells per well) and cultured for 18 hours. After incubation, L. johnsonii cells were removed by centrifugation at 4,000 × g for 10 minutes at 4°C, followed by filtration of the supernatant through a 0.22 μm membrane filter to obtain the conditioned medium. The conditioned medium was then diluted 1:1 with fresh complete IMDM and supplemented with IL-23 (final pH ≈ 5.5) before use in iTh17 cell cultures. For neutralized conditioned medium, NaOH was added to the diluted conditioned medium to adjust the pH to 7 prior to culturing iTh17 cells.
RNA extraction and quantitative PCR analysis
Total RNA was isolated from iTh17 cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific), following the manufacturer’s protocol. The concentration and purity of the extracted RNA were measured using a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific). Gene expression levels were quantified by reverse transcription quantitative PCR (RT-qPCR) using the Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) on a QuantStudio 3 Real-Time PCR System (Applied Biosystems). The primer and probe sequences used for detection were as follows: for Rpl38 mRNA, forward primer: 5’-GCGGAAGGATGCCAAGT-3’, reverse primer: 5’-GTGATAACCAGGGTGTAAAGGT-3’, and probe: 5’-ATGTGAAGTTCAAGGTTCGCTGCAG-3’; for II17a mRNA, forward primer: 5’-CCGCAATGAAGACCCTGATAG-3’, reverse primer: 5’-GCTTTCCCTCCGCATTGA-3’, and probe: 5’-TGGGAAGCTCAGTGCCGCCAC-3’; for Rorc mRNA, forward primer: 5’-CCGCTGAGAGGGCTTCAC-3’, reverse primer: 5’-TGCAGGAGTAGGCCACATTACA-3’, and probe: 5’-AAGGGCTTCTTCCGCCGCAGCCAGCAG-3’.
Fluorescence microscopy
Differentiated iTh17 cells were cultured on coverslips in 12-well plates and subjected to the designated treatments. After treatment, the plates were centrifuged at 1,200 rpm for 2 minutes, and the culture medium was carefully removed. Cells were then fixed and permeabilized using the Foxp3 staining buffer set (eBioscience) for 30 minutes at room temperature. Subsequently, cells were stained sequentially with a rabbit anti-IL-17A polyclonal antibody (Cat# PA5–114455, Invitrogen) followed by a donkey anti-rabbit IgG antibody conjugated to Alexa Fluor 488 (Cat# A5–21206, Invitrogen). Nuclei were visualized by mounting the coverslips with ProLong Gold Antifade Mountant containing DAPI (Invitrogen). Fluorescence images were acquired using a ZEISS Axioplan 2 microscope.
Flow cytometry
iTh17 cells were differentiated in 96-well plates (Fisher Brand) at a density of 2 × 10^5^ cells per well and subjected to the designated treatments. After treatment, plates were centrifuged at 1,200 rpm for 2 minutes, and the culture medium was carefully removed. Cells were first blocked with anti-CD16/CD32 antibodies (Fc block; BD Pharmingen), followed by cell surface staining with fluorochrome-conjugated anti-mouse CD4 antibody (clone GK1.5, BioLegend). Subsequently, the cells were fixed and permeabilized using the Foxp3 staining buffer set (eBioscience), and then stained for intracellular RORγT (clone Q31–378, BD Pharmingen) and IL-17A (clone TC11–18H10.1, BioLegend). Flow cytometric analysis was performed using a Cytek Aurora analyzer (Cytek Biosciences). Data were analyzed using FlowJo software, version 10.9.0 (FlowJo, LLC).
Labeling cell newly synthesized proteins with βES
iTh17 cells were differentiated in 96-well plates at a density of 2 × 10^5^ cells per well and subjected to the designated treatments. Cell cultures were supplemented with 0.4 mM β-ethynylserine (βES), generously provided by Dr. Kimberly M. Bonger (Radboud University, the Netherlands), for 30 minutes. Cells treated with 100 μM cycloheximide (Fisher Scientific) served as negative controls for protein synthesis. Following βES incubation, cells were chased with complete IMDM for 1 hour at 37°C.
Subsequently, cells were fixed and permeabilized using the Foxp3 Staining Buffer Set (eBioscience) for 15 minutes at room temperature to facilitate the azide-alkyne click reaction and subsequent flow cytometry analysis. Copper-catalyzed azide-alkyne click (CuAAC) chemistry was performed using the Click-iT^™^ Cell Reaction Buffer Kit and Alexa Fluor^™^ 488 Azide (Invitrogen). The reaction buffer cocktail was prepared following the manufacturer’s instructions, and 100 μL was added to each well. The plates were then incubated at room temperature for 30 minutes. After the click reaction, cells were stained for CD4, IL-17A, and RORγt for flow cytometric analysis as above.
ELISA
The concentrations of mouse IL-17A and IL-10 in culture supernatants were measured using enzyme-linked immunosorbent assay (ELISA) DuoSet kits (R&D Systems), according to the manufacturer’s instructions. Absorbance was measured at 450 nm (with wavelength correction at 570 nm) using a BioTek Synergy H1 microplate reader (Agilent Technologies).
Statistical analysis
Statistical analyses were conducted using GraphPad Prism version 9.0 (GraphPad Software). For comparisons between two groups, significance was assessed using 2-tailed Student’s t-tests for data with a normal distribution. When comparing three or more groups, one-way ANOVA followed by Tukey’s multiple comparisons test was employed to evaluate the significance between groups.
Supplementary Material
1
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