MicroRNA-98-5p Inhibits IFI44L-Mediated Differentiation of Dendritic Cells and Activation of Interferon Pathway in Systemic Lupus Erythematosus

ABSTRACT MicroRNA-98-5p (miR-98-5p) plays a protective role in the pathogenesis of autoimmune diseases through anti-inflammatory effects, but little is known about its role in Systemic lupus erythematosus (SLE). Our previous study suggested Interferon-inducible 44 like (IFI44L) overexpressed in monocytes which contributes to the pathogenesis of SLE by enhancing the maturation and functions of monocyte-derived dendritic cells (Mo-DCs), and miR-98-5p can regulate the expression of IFI44L. In this study, we identified miR-98-5p lowly expressed in both peripheral blood mononuclear cells (PBMCs) and monocytes of SLE patients along with high expression of IFI44L. IFI44L serves as target gene of miR-98-5p which inhibits differentiation of Mo-DCs and IFI44L-mediated activation of interferon pathway. We further showed that miR-98-5p promotes methylation of the IFI44L promoter to down-regulate its expression in SLE. Our results reveal an important role for miR-98-5p in the IFI44L-mediated immune imbalance of SLE and suggest a potential therapeutic target for SLE in the future.


Introduction
Systemic lupus erythematosus (SLE) is an extremely complex autoimmune disease caused by autoimmune reaction, with autoimmune inflammation as the prominent manifestation and multiple system damage (Durcan et al., 2019;Tsokos, 2011).Studies have shown that the dysfunction of immune cells and the destruction of immune homeostasis are important causes of the development of SLE (Akhil et al., 2023;Lazar & Kahlenberg, 2023).Abnormal proliferation and activation of monocytes and dendritic cells (DCs) play important roles in SLE pathogenesis.The inflammatory cascade in SLE is triggered through DCs and then produces cytokines such as IL-1β, IL-4, IL-6, IL-17, and TGF-β, which activate and polarize auto-aggressive helper T cells toward different cell subsets (Aringer, 2020;Luo et al., 2022).Thus, further elucidation of monocyte subset dysfunction and the molecular mechanisms in SLE are ongoing hotspot investigations.MicroRNAs (miR) have a wide range of biological effects, which can not only be used as a marker to assess the condition but also as a target to study the pathogenesis and prevention measures (Lu & Rothenberg, 2018;Wu et al., 2020).Previous studies suggested that miR-98-5p plays a protective role in the pathogenesis of immune diseases through anti-inflammatory effects and is downregulated in several autoimmune diseases (Liu et al., 2018;Wang et al., 2021;Yu et al., 2021).Our studies have found that interferon-related genes (including IFI44L, IFIT1, MX1, STAT1) are at low methylation levels in T cells of SLE patients (He et al., 2022).Afterward, two methylation sites in the promoter region of IFI44L gene were found in the peripheral blood of SLE patients using DNA methylation chip, which can be used as biomarkers for the auxiliary diagnosis of SLE (Zhao et al., 2016).In this study, we investigated the expression of miR-98-5p and IFI44L in monocytes and DCs from SLE patients and analyzed inhibitory effects of miR-98-5p on IFI44L-mediated differentiation of DCs and activation of interferon pathway.Moreover, we also revealed that the decreased expression of IFI44L was inhibited by miR-98-5p through epigenetic mechanisms.

SLE patients and healthy controls
A total of 30 SLE patients (mean age 31.14 ± 3.48 years old) were recruited from The Second Xiangya Hospital of Central South University.All patients fulfilled the 2019 European League Against Rheumatism/American College of Rheumatology Classification Criteria (EULAR/ACR) for SLE (Aringer et al., 2019) and Disease activity was assessed by the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) (Gladman et al., 2002) at the time of blood collection.Patients' information is shown in Supplemental Table A1.A total of 30 healthy controls (mean age 33.790 ± 3.84 years) were recruited from Changsha Blood Center without any autoimmune disease history.All SLE patients and healthy controls were matched for age and sex in all experiments.Our human sample study was under a protocol approved by the Ethics Committee of The Second Xiangya Hospital of Central South University.Written informed consent was obtained from all subjects.

Cell isolation and culture
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Sweden).CD14 + monocytes and naive CD4 + T cells were isolated from PBMCs using magnetic beads (Human CD14 Monocyte Cocktail Kit, Human naïve CD4 T Cell Isolation Kit II (Miltenyi Biotec, Germany) according to the manufacturer's instructions.The purity of the sorted cells was greater than 90%.CD14 + monocytes and naive CD4 + T cells were cultured in RPMI 1640 (Gibco, China) medium supplemented with 10% FBS (HyClone, China), 100 U of penicillin, and 100 mg of streptomycin at 37°C in a 5% CO 2 atmosphere.

In vitro cell differentiation
In the cell differentiation experiment, Mo-DCs were differentiated from naïve CD14 + monocytes.CD14 + monocytes were separated from the peripheral blood according to the above methods.Purified naïve CD14 + monocytes were stimulated with GM-CSF (20 ng/mL; PeproTech, USA) and IL-4 (20 ng/mL; PeproTech, USA) for 7 days, followed by treatment with LPS (20 ng/mL; Sigma, USA) for 3 days.The treated cells were harvested for subsequent experiments.

RNA isolation and quantitative PCR (RT-qPCR)
Total RNA was extracted using Trizol reagent (Invitrogen, USA).Expression levels of miR-98-5p were quantified by using the TaqMan microRNA assay specific for miR-98-5p (Applied Biosystems, USA) according to the manufacturer's instructions.U6-snRNA was used as an endogenous control.For quantification of other mRNA, cDNA was synthesized by PrimeScript® RT reagent kit with gDNA Eraser (TaKaRa, China) using 1 μg of total RNA according to the manufacturer's instructions.Transcripts were measured using a Roche-LightCycler96 Real-Time PCR System (Basel, Switzerland).The value of each cDNA was calculated using the 2 −ΔCt method: ΔCycle threshold (CT) = CT IFI44L − CT GAPDH , and normalized to GAPDH.All primer sequences are listed in Supplemental Table A2.

Luciferase assay
For 3′-UTR reporter assays, the 3′-UTRs of the IFI44L mRNA containing WT or MU miR-98-5p binding sites were subcloned into the psiCHECK-2 vector (Promega, USA).Then, 1 × 10 5 HEK293T cells were seeded in the wells of a 24-well plate 1 day before transfection, and each well was transfected with a mixture of 10 ng 3′-UTR luciferase reporter vector (WT or MU) and agomir-98-5p (sequence, UGAGG UAGUA AGUUG UAUUG UU) or agomir control (sequence, UCACA ACCUC CUAGA AAGAG UAGA) using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions.Cells were lysed after 24 hours and luciferase activity was detected using the Dual-Luciferase Reporter Assay System (Promega, USA).The ratio of Renilla luciferase activity to firefly luciferase activity was calculated for each well.

Western blot (WB)
Cells were lysed in protein lysis buffer and quantified by the Bradford assay (HyClone-Pierce, USA) followed by 8% vertical dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, USA).The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer and then incubated with primary antibodies including rabbit anti-human IFI44L (Abcam, UK) or mouse anti-human GAPDH (Abcam, UK), followed by HRP-goat anti-rabbit IgG antibody (Abcam, UK).Proteins were detected with an ECL Western blot detection kit (Thermo Scientific, USA).Quantification of IFI44L was normalized to GAPDH by densitometry.

Flow cytometry
Surface markers were evaluated using FACSCanto II (BD Biosciences).Firstly, cells were incubated with FcR blocking reagent (Miltenyi, Germany) for 10 min followed by primary antibodies on ice in the dark for 30 min.The antibodies used for surface marker analysis included FITC anti-CD40, PE anti-CD80, PE anti-CD83, and FITC anti-CD86 (BD Biosciences, USA).Data was analyzed using FlowJo (Tree Star, USA).

Statistical analysis
All quantitative data were presented as means ± SEM.Statistical significance was assessed by t-test (two-tailed).Correlation analysis was assessed by Spearman's correlation coefficient.All data were calculated in SPSS 23.0 software.

miR-98-5p expression decrease in SLE monocytes
To explore the role of miR-98-5p in SLE monocytes, we first detected the expression levels of miR-98-5p in PBMCs and CD14 + monocytes of SLE patients and healthy controls using RT-qPCR.The results revealed the expression levels of miR-98-5p in monocytes were significantly decreased in SLE patients than in healthy controls (Figure 1a,b).Notably, we observed the expression levels of miR-98-5p in both PBMCs and CD14 + monocytes from SLE patients were negatively correlated with the SLEDAI scores (Figure 1c,d).In addition, we established IFN-α and SLE serum-treated CD14 + monocytes, which closely resemble the human disease phenotype according to our previously published studies (Luo et al., 2022).As expected, the results revealed the expression levels of miR-98-5p in IFN-α and SLE serum-treated CD14 + monocytes were all significantly decreased in SLE patients than in untreated controls (Figure 1e,f).These data demonstrated that the abnormal miR-98-5p expression may be functionally involved in the CD14 + monocyte -mediated immune dysfunction in SLE.

IFI44L expression increase in SLE monocytes
To explore the relationship between miR-98-5p and IFI44L, we also measured the expression levels of IFI44L in monocytes of SLE patients and healthy controls using RT-qPCR.The results revealed the expression levels of IFI44L mRNA in PBMCs and CD14 + monocytes were significantly increased in SLE patients than in healthy controls (Figure 2a,b).Similarly, we observed the expression levels of IFI44L mRNA in both PBMCs and CD14 + monocytes from SLE patients were positively correlated with the SLEDAI scores (Figure 2c,d).In addition, we also observed the expression levels of IFI44L in both PBMCs and CD14 + monocytes from SLE patients were negatively correlated with the expression levels of miR-98-5p mRNA (Figure 2e,f).These results demonstrated that upregulation of IFI44L promotes and aggravates the development of SLE, but miR-98-5p may inhibit this effect.

IFI44L is a target gene of miR-98-5p
To gain insight into the molecular mechanisms of how miR-98-5p regulates IFI44L expression in SLE, we used the bioinformatics tool of TargetScan (http://www.targetscan.org)to predict the putative targets of miR-98-5p.The results showed the binding sites between miR-98-5p and IFI44L mRNA (Figure 3a).To validate this target site, we generated WT luciferase reporter constructs that included the 3′ untranslated region (UTR) of the IFI44L genes and mutant type (MU) reporter constructs, which contained mutant binding sequences of miR-98-5p (Figure 3a).Compared with the negative control agomir (agomir-NC) transfected cells, cells transfected with agomir-98-5p showed significantly decreased luciferase activity with the WT constructs, but this inhibition was lost with transfection of the MU construct (Figure 3b).
We next evaluated whether miR-98-5p influences endogenous IFI44L protein levels.Actually, we found that the protein levels of IFI44L were significantly decreased by transfection with agomir-210 but were significantly increased after transfection with antagomir-210 compared with their corresponding controls (Figure 3c).In addition, we also performed gene recovery experiments.Firstly, cells were transfected with agomir-98-5p and agomir-NC.Notably, we found that transfection of IFI44L overexpression plasmid could recover the low expression levels of IFI44L induced by miR-98-5p (Figure 3d,e).Together, these data from luciferase reporter assay and transfection experiments indicated that IFI44L is a direct target of miR-98-5p.

miR-98-5p promotes methylation of IFI44L
Our previous results indicated the important role of IFI44L demethylation in SLE pathogenesis.Therefore, we sought to evaluate the potential efficacy of miR-98-5p on methylation modification of IFI44L.To further clarify the role of miR-98-5p in IFI44L expression, we transfected agomir-98-5p and agomir-NC into normal monocytes and detected the methylation levels of two CpG sites using BSP.The results showed that DNA methylation levels of IFI44L promoter were significantly increased in monocytes transfected with agomir-98-5p than in agomir-NC (Figure 6a,b).These data indicated that miR-98-5p could induce epigenetic modification of IFI44L to regulate its expression in SLE.

Discussion
Numerous studies have revealed that interferon pathways and microRNAs are frequently involved in regulating immune cell development, differentiation, and immune responses in SLE (Le et al., 2017;Mirzaei et al., 2021;So et al., 2021;Stypinska & Paradowska-Gorycka, 2015;Wang et al., 2021;Zan et al., 2014).In this study, we showed that miR-98-5p is downregulated in PBMCs and CD14 + monocytes from SLE patients.On the contrary, IFI44L is significantly downregulated in PBMCs and CD14 + monocytes from SLE patients.In addition, we analyzed the correlation between miR-98-5p expression and IFI44L expression in both PBMCs and CD14 + monocytes from SLE patients, which showed a significantly negative correction.The results suggested that miR-98-5p has apparent anti-inflammatory effects, but in SLE these anti-inflammatory effects weakened and induced increase of IFI44L.Abnormal miR-98-5p expression functionally involved in the IFI44L -mediated immune dysfunction in SLE.
A miRNA may have multiple target mRNAs and only a small number of key target genes mediate its function (Lu & Rothenberg, 2018;Wu et al., 2020).In previous studies, miR-98-5p was found to negatively modulate IL-6 expression and  may be a potential clinical approach in rheumatoid arthritis (RA) (Wang et al., 2021).CKIP-1 was a target gene of miR-98-5p and miR-98-5p regulated osteoblast differentiation in MC3T3-E1 cells by targeting CKIP-1 (Liu et al., 2018).In addition, miR-98-5p was identified as a candidate predictor of liver fibrosis progression.miR-98-5p is reduced in activated L×2cells, and miR-98-5p overexpression inhibited the hepatic stellate cells (HSCs) activation (Ma et al., 2022).miR-98-5p could protect against oxidative through downregulating the level of BTB domain and CNC homology 1 and upregulating the levels of NAD(P)H: quinone oxidoreductase 1 and heme oxygenase 1 (Yu et al., 2021).To the best of our knowledge, previous studies have not found a relationship between IFI44L and miR-98-5p.In order to verify the relationship between IFI44L and miR-98-5p, we predicted the binding sites between IFI44L and miR-98-5p, and perform the luciferase reporter assay and transfection experiments.As expected, our results all supported the prediction that IFI44L is a direct target of miR-98-5p.
Immature DCs differentiate into mature antigen-presenting DCs is associated with high expression of costimulatory receptors on DCs (Balan et al., 2019;He et al., 2019;Reis e Sousa, 2006;Yin et al., 2021).CD40, CD80, CD83, and CD86 are usually known as markers for mature DCs and costimulatory receptors (Shenoda & Ajit, 2016;Tekguc et al., 2021;Wardowska, 2020).Our previous study suggested that aberrant activation of Mo-DCs was involved in the pathogenesis of SLE (Luo et al., 2022).In the present study, we changed the miR-98-5p levels in naïve monocytes and induced them differentiating into Mo-DCs to simulate the changes in SLE.Our results demonstrated a lower CD40, CD80, CD83, and CD86 expression on Mo-DCs, suggesting an abnormal downregulation of maturation and immune activity in SLE induced by miRNA-98-5p.These results suggested that miR-98-5p was a potential anti-inflammatory factor and partly explained its abilities to inhibit autoimmune responses.
Proinflammatory cytokine stimulation of interferon pathway is a key initiating factor for aggravation of SLE (Aringer & Smolen, 2005;Thanou et al., 2021).Previous studies showed that interferon pathway associated genes are increased in serum and PBMCs of SLE (Aringer, 2020;  Stypinska & Paradowska-Gorycka, 2015).In this study, both knockdown and overexpression experiments indicated IFI44L promoted IL-1β, IL-4, IL-6, and TGF-β expression.However, IL-17A didn't show any different in our experiments, which indicates that IL-7 does not belong to the interferon pathway and is not affected by miR-98-5p.Consistently, we also found that miR-98-5p regulates these cytokine expressions and overexpression of IFI44L partly abolished the regulatory effects of miR-98-5p on cytokine expression.IFI44L has been reported as an important initiation and effector factor in interferon pathway (Luo et al., 2022;Zhao et al., 2016Zhao et al., , 2021)).Consequently, our findings highlight an important role for miR-98-5p as an antiinflammatory factor and inhibit IFI44L-mediated activation of the interferon pathway in SLE.
Epigenetic mechanisms underlying overexpression of IFI44L in SLE were still unknown (Akhil et al., 2023;Wu et al., 2020).Our previous studies revealed TET2 recruited by STAT3 induces DNA demethylation of IFI44L promoter (Luo et al., 2022).Thus, we also link the miR-98-5p with methylation of IFI44L in the present study.The results showed that DNA methylation levels of IFI44L promoter were significantly increased when miR-98-5p was overexpressed.Our results suggested again miR-98-5p is a protective miRNA in immune responses and further clarify the upstream regulation mechanism of IFI44L abnormal expression in SLE.In addition, there are few literatures reported miR-98-5p mimic significantly reduced the expression of STAT3 (Dong et al., 2020;Guo et al., 2023).Even if these results hold true, they are still consistent with our previous results.For more accurate instructions, we also tested the corresponding results and found no significant change in STAT3 expression when transfected with miR-98-5p mimic (Supplemental Figure 1).
In conclusion, the present study identifies miR-98-5p is lowly expressed in SLE patients along with high expression of IFI44L.IFI44L is a target gene of miR-98-5p which inhibits differentiation of Mo-DCs and IFI44L -mediated interferon response through repressing IFI44L expression, contributing to several aspects of the immune imbalance in SLE.Moreover, miR-98-5p also promotes methylation of IFI44L to regulate its expression in SLE.In the future, we will continue our research to test this finding in Rheumatic arthritis (RA) and other autoimmune diseases.Our study firstly demonstrates the role of miR-98-5p in pathogenesis of SLE, which was not shown in other studies.These results may help us identify a novel miRNA and provide new insights into therapeutic strategies for SLE.

Figure 1 .
Figure 1.miR-98-5p expression decrease in SLE monocytes.(a, b) The expression level of miR-98-5p in PBMCs (a) and CD14 + monocytes (b) from 30 SLE patients and 30 healthy controls were measured by RT-qPCR.(c, d) Correlation between SLEDAI and miR-98-5p expression level in PBMCs (c) and CD14 + monocytes (d) from 30 SLE patients.(e, f) The expression levels of miR-98-5p in IFN-α (e) and SLE serum (f) treated CD14 + monocytes were measured by RT-qPCR.****P < .0001.Data are shown as mean ± SEMs.Student's t-test was used to compare the results.Spearman's correlation coefficient was used for the correlation analysis.

Figure 2 .
Figure 2. IFI44L expression increase in SLE monocytes.(a, b) The expression level of IFI44L mRNA in PBMCs (a) and CD14 + monocytes (b) from 30 SLE patients and 30 healthy controls were measured by RT-qPCR.(c, d) Correlation between SLEDAI and IFI44L mRNA level in PBMCs (c) and CD14 + monocytes (d) from 30 SLE patients.(e, f) Correlation between miR-98-5p expression level and IFI44L mRNA level in PBMCs (e) and CD14 + monocytes (f) from 30 SLE patients.Data are shown as mean ± SEMs.Student's t-test was used to compare the results.Spearman's correlation coefficient was used for the correlation analysis.****P < .0001.

Figure 3 .
Figure 3. IFI44L is downstream target of miR-98-5p.(a) miR-98-5p target sequences (in red) of the IFI44L mRNAs and corresponding mutant sequences, which were included in luciferase reporter vectors.(b) Luciferase activities were measured in HEK293T cells cotransfected with luciferase reporter vectors and agomir-98-5p or agomir-NC, n = 10 per group.(c) HEK293T cells were transfected with agomir-98-5p or agomir-NC and antagomir-98-5p or antagomir-NC.IFI44L protein levels were analyzed by WB. (d, e) HEK293T cells were transfected with agomir-98-5p or agomir-NC, and after 24 hours, the IFI44L overexpression plasmid or plasmid control (n = 10) were transfected into cells.Then, cells were collected to detect the protein levels (d) and mRNA levels (e) of IFI44L at 48 hours after transfection.Data are shown as mean ± SEMs.Student's t-test was used to compare the results.*P < .05. ns, not significant.

Figure 6 .
Figure 6.miR-98-5p promotes methylation of IFI44L promoter.(a) It shows the position of two methylation sites within the IFI44L promoter region in chromosome 1; (b) Mo-DCs induced from normal CD14 + monocytes were transfected with agomir-98-5p or agomir-NC, and after 24 hours, the methylation levels of two site in IFI44L promoter were detected by BSP.Data are shown as mean ± SEM.Student's t-test was used to compare the results.*P < .05.**P < .01.