sNASP Mutation Aggravates to the TLR4-Mediated Inflammation in SLE by TAK1 Pathway

Genetic factors play an important role in the pathogenesis of systemic lupus erythematosus (SLE), and abnormal Toll-like receptor (TLR) signaling pathways are closely related to the onset of SLE. In previous studies, we found that the mutant somatic nuclear autoantigenic sperm protein (sNASP) gene in the mouse lupus susceptibility locus Sle2 can promote the development of lupus model mice, but the mechanism is still unclear. Here, we stimulated mouse peritoneal macrophages with different concentrations of lipopolysaccharide. The results showed that sNASP gene mutations can promote the response of the TLR4–TAK1 signaling pathway but have no significant effect on the TLR4–TBK1 signaling pathway. sNASP mutations enhanced TLR4-mediated nuclear factor-κ-gene binding and mitogen-activated protein kinase activation and IL-6, tumor necrosis factor secretion in murine peritoneal macrophages. Collectively, our study revealed the impact of sNASP gene mutation on the sensitivity of TLR4 receptors in mouse peritoneal macrophages and shed light on potential mechanisms underlying inflammation in autoimmune diseases.


Introduction
Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease characterized by the production of autoantibodies and subsequent inflammation that affects multiple organs [1,2].The etiology of SLE involves a complex interplay between genetic and environmental factors.While genomewide association studies have successfully identified more than 100 susceptibility gene loci associated with SLE [3], the specific genes responsible for its clinical pathogenicity remain to be elucidated.
Mouse models have significantly contributed to our understanding of the pathogenesis of SLE [4].In the NZM2410 model, three susceptibility loci associated with lupus, namely Sle1, Sle2, and Sle3, have been identified [5].In our previous study, we utilized whole exome sequencing to identify a variant of somatic nuclear autoantigenic sperm protein (sNASP) from the Sle2 locus.This variant harbored two mutations in exon 10, specifically 841G>A and 844C>T in the sNASP cDNA sequence, resulting in the substitution of two consecutive amino acid residues (V281I and L282F) within the sNASP protein.Based on these findings, we hypothesized that the mutated sNASP represents a pathogenic gene at the Sle2 locus.To validate this hypothesis, we introduced the mutated sNASP into B6.lprmice, generating B6.ΔsNASP.lprmice.In comparison to the control B6.lpr mice, the B6.ΔsNASP.lprmice exhibited enlarged spleen and lymph nodes, elevated total counts of T and B cells, increased activation and effector T cells, heightened levels of autoantibodies, and significantly enhanced inflammation in the kidneys and lungs [6].Furthermore, we introduced the sNASP mutation into Sle1.Yaa mice, which demonstrated higher proportions of CD3 + T cells and activated CD19 + CD86 + B cells in the spleen and lymph nodes compared to Sle1.Yaa mice [7].
Numerous studies have highlighted the role of sNASP, a histone chaperone protein, in the storage and transportation of histones, particularly histones H1, H3, and H4.sNASP is involved in regulating the dynamic balance of nucleosome formation, chromatin assembly, and disassembly, thereby influencing the conformation and accessibility of chromatin [8,9].
Importantly, sNASP has the ability to bind to TRAF6 in unstimulated macrophages and prevent its own ubiquitination and subsequent degradation, thus exerting a negative regulatory effect on the Toll-like receptor (TLR) signaling pathway [10].Recent studies have observed elevated mRNA expression levels of TLR4, TLR7, and TLR9 in SLE patients compared to healthy individuals [11,12].In vitro experiments have shown that mutations in TLR7 lead to increased production of inflammatory factors and elevated autoantibody titers [13], suggesting a crucial role for TLRs in the pathogenesis of SLE.
The impact of sNASP gene mutation on the TLR signaling pathway remains poorly understood.In this study, we employed various concentrations of lipopolysaccharide (LPS) to stimulate mouse peritoneal macrophages, aiming to elucidate the effects of sNASP gene mutations on the TLR4 signaling pathway and the subsequent production of inflammatory cytokines.Our research findings are expected to offer novel insights into the mechanistic role of sNASP mutation in promoting SLE in mice.

Material and Methods
2.1.Mice.Cyagen Biosciences Inc. (Guangzhou, Guangdong, China) generated the B6.ΔsNASP mouse by introducing mutated bases of the sNASP allele into the C57BL/6J (B6) genome [6].Briefly, two homology arms were made through PCR using BAC clones RP24-384F21 and RP24-72F14 from the B6 library as a template.The CTGTACTCCATGAGC sequence in exon 10 of the sNASP gene was edited to CTATATTCCATGAGC in the 5′ homology arm.The constructed targeting vector was electroporated into embryonic stem (ES) cells of C57BL/6 mouse, and then selected positive ES clones were microinjected into blastocysts.After genotyping, chimeric mice were bred with an Flp-deleter mouse to create an F1 mouse through Flpmediated recombination.Finally, F1 mice intercrossed to obtain a homozygous B6.ΔsNASP model.The B6.ΔsNASP.lpr line was derived by breeding male B6.ΔsNASP mice-tofemale B6.lpr mice and subsequent intercrossing of progeny.B6.lpr was purchased from the Jackson Laboratory (Bar Harbor, ME, USA).B6.WT (C57BL/6) mice were provided by the Experimental Animal Center of Weifang Medical University.Female mice aged 3 months were used in this experiment.All animals were cared for under experimental protocols approved by the Weifang Medical University Animal Care Committee and housed in a specific pathogen-free facility.

Acquisition of Peritoneal Macrophages and LPS
Stimulation.To obtain mouse primary peritoneal macrophages, a mouse was euthanized using carbon dioxide and sprayed with 75% ethanol.Scissors were used to expose the inner skin lining the peritoneal cavity, following which peritoneal lavage was performed using phosphate buffer saline with 3% fetal calf serum.Samples were then centrifuged and reconstituted into fresh culture media.The cells were next transferred to a 6-well plate with the number of 1 × 10 6 cells per well and incubated for 3 hr.After incubation, the media was changed to remove free-floating cells.Three hours later, nonadherent cells were removed, and the adherent monolayer cells were used as peritoneal macrophages [14].The cells were cultured at 37°C under 5% CO 2 in RPMI 1640 supplemented with 12% fetal bovine serum.After peritoneal macrophages were stimulated with LPS (1, 10, 100, 0/0.5/1/2 hr), the supernatants of cell culture were collected for the detection of cytokines, and the cell precipitates were collected for quantitative polymerase chain reaction (qPCR) and Western blot detection.
2.5.Western Blot.For immunoblot analysis, cells were lysed with Servicebio (Wuhan, China) RIPA Lysis Buffer (catalog number: G2002) supplemented with a protease inhibitor mixture (catalog number: G2007; Servicebio), and then the protein concentrations of the intermixture were measured with a bicinchoninic acid assay (catalog number: P0012; Beyotime).Equal amounts of extracts were separated by SDS-PAGE and then were transferred onto polyvinylidene fluoride membranes for immunoblot analysis.

Real-Time Reverse Transcription-Polymerase Chain
Reaction.RNA was extracted from cells using the RNAex pro reagent (code no: AG21101-S, Accurate Biotechnology, Hunan, China) and converted into cDNA by reverse transcription using the Evo M-MLV RT Premix Kit (code no: AG11706, Accurate Biotechnology, Hunan, China).The sequences of primers were (forward) 5′-CAGGCGGTGCCTATGTCTC-3′ and (reverse) 5′-CGATCACCCCGAAGTTCAGTAG-3′ for TNF-α, (forward) 5′-TGTGCAATGGGCAATTCTGAT-3′ and (reverse) 5′-GGTACTCCAGAAGACCAGAGGA-3′ for IL-6 and (forward) 5′-TCTTTGCAGCTCCTTCGTTGCCGGTCC-3′ and (reverse) 5′-GTCCTTCTGACCCATTCCCACCATC-CAC-3′ for β-actin.[15,16].Excessive expression of inflammatory factors exacerbates the inflammatory response [17].In order to examine the potential inhibitory function of sNASP and whether the mutation of the sNASP gene could enhance the activation of the TLR4 receptor, resulting in the expression of inflammatory cytokines, we assessed the levels of IL-6 and TNF-α proteins and mRNAs in mouse peritoneal macrophages after stimulation with different concentrations of LPS using ELISA and qPCR.Following various levels of LPS stimulation, both the mRNA and protein levels of IL-6 in peritoneal macrophages from B6. ΔsNASP.lprmice were higher compared to those from B6.lpr mice (Figure 1(a)-1(f)).Similarly, the levels of TNF-α mRNA and protein were also increased (Figure 1(g)-1(l)).Additionally, the expression levels of IL-6 and TNF-α mRNAs and proteins in peritoneal macrophages from B6.ΔsNASP mice were significantly higher than those from B6.WT mice following different concentrations of LPS stimulation (Figure 2(a)-2(l)).Taken together, these findings suggest that mutations in the sNASP gene can impact the sensitivity of the TLR4 receptor, leading to increased expression levels of IL-6 and TNF-α genes and proteins in mouse peritoneal macrophages.

sNASP Mutation Downregulates Protein Expression of TRAF6
. LPS, as a specific agonist for TLR4, can effectively activates sNASP, leading to its dissociation from the TRAF6 complex and subsequent ubiquitination and degradation of TRAF6.This activation triggers downstream signaling pathways and induces the release of inflammatory factors from the cells [10,18].To assess the expression of sNASP and TRAF6, we initially stimulated peritoneal macrophages obtained from B6.lpr mice, B6.ΔNASP.lprmice, B6.WT mice, and B6.ΔNASP mice with different concentrations of LPS (1, 10, 100 ng/ml) and quantified the levels of sNASP and TRAF6 proteins.Interestingly, we did not observe any significant differences in the protein levels of sNASP in peritoneal macrophages among B6.ΔNASP.lpr,B6.lpr, and B6.ΔNASP mice compared to the B6.WT control (Figure 3(a)-3(f )).However, the expression of TRAF6 following LPS stimulation showed a significant reduction.This reduction in TRAF6 expression may indicate a change in the sensitivity of the TLR4 receptor, particularly evident at 1 hr post-LPS stimulation.These findings suggest that the sNASP mutation does not affect the quantity of sNASP but may influence its function, potentially contributing to TRAF6 degradation and subsequent activation of the downstream signaling pathway.

sNASP Mutation
Promotes TLR4-Induced NF-κB and MAPK Activation.The activation of TLR4 involves two downstream signaling pathways, namely the TLR4-TAK1 pathway and the TLR4-TBK1 pathway.Here, mouse models (B6.lpr,B6.ΔNASP.lpr,B6.WT, and B6.ΔNASP) and western blot were used to investigate the protein dynamics alterations involved in TLR4-TAK1 pathway that is potentially under the modulation by TRAF6.Mouse peritoneal macrophages were stimulated with 1 ng/ml LPS for durations of 0.5, 1, and 2 hr.The levels of p-TAK1 and p-p38 in peritoneal macrophages of B6.ΔNASP.lprmice were higher than those in B6.lpr mice only after 2 hr of stimulation (Figure 4(a)).In the groups stimulated with 10 ng/ml and 100 ng/ml LPS, the levels of p-TAK1, p-p65, and p-p38 in peritoneal macrophages of B6.ΔNASP.lprmice were significantly higher than those in B6.lpr mice at 0.5 hr after LPS stimulation.There was no significant change in p-JNK protein (Figures 4(b) and 4(c)).These findings indicate that mutations in the sNASP gene increase the protein expression levels of the TLR4-TAK1 signaling pathway in mouse peritoneal macrophages compared to B6.lpr mice.In summary, the mutation in the sNASP gene enhances the sensitivity of the TLR4 receptor in B6.lpr mice, resulting in the activation of nuclear factor-κ-gene binding (NF-κB) and mitogen-activated protein kinase (MAPK) pathways.As Fas ligands have been shown to promote a TLRdependent model of lupus-like inflammation [19], we sought to rule out the effect of Fas deficiency on TLR4-TAK1 pathway.The expression of p-TAK1, p-p65, p-JNK, p-p38, and p-IκBα in peritoneal macrophages from both B6.WT mice and B6.ΔNASP mice following stimulation with different concentrations of LPS under identical conditions were examined to test the hypothetical effects of Fas deficiency.After being stimulated with 1 ng/ml LPS for 2 hr, the expression levels of p-TAK1, p-p65, p-JNK, and p-p38 proteins in peritoneal macrophages from B6.ΔNASP mice were higher compared to those from B6.WT mice (Figure 5(a)).Furthermore, the levels of p-TAK1, p-p65, and p-p38 in peritoneal macrophages from B6.ΔNASP mice were significantly higher than those from B6.WT mice at 0.5 hr following stimulation with 10 and 100 ng/ml LPS (Figures 5(b) and 5(c)).Additionally, the expression level of p-JNK protein was also higher in B6.ΔNASP mice compared to B6.WT mice at 0.5 hr after stimulation with 10 ng/ml LPS, but there was no difference observed at 100 ng/ml LPS.Overall, these observations suggest that mutations in the sNASP gene indeed lead to increased protein expression levels of the TLR4 TAK1 signaling pathway in mouse peritoneal macrophages, thereby enhancing the activation of the TLR4 receptor through the TLR4 TAK1 signaling pathway in response to LPS stimulation.Notably, a comparison between Figures 2 and 3 reveals distinct variations in p-p65 and p-TAK1 alterations at the lowest LPS concentration, implying an influence of the lpr mutation.In conclusion, the sNASP gene mutation in B6.WT mice genuinely improves the sensitivity of the TLR4 receptor compared to lpr mice, resulting in the activation of the NF-κB and MAPK pathways independent of Fas.

sNASP Mutation Does Not Impact TLR4-Induced IRF3
Activation.To investigate the impact of mutation on the TLR4-TBK1 signaling pathway, we examined the protein expression levels of this pathway in mouse peritoneal macrophages after LPS stimulation using western blot analysis.ΔsNASP mice, the expression levels of TBK1 and phosphorylated IRF3 proteins in the TLR4-TBK1 signaling pathway did not show significant differences among the groups when stimulated with various concentrations of LPS (Figure 6(a)-6(f)).These findings indicate that there were no notable changes in protein levels within the TLR4-TBK1 signaling pathway following LPS stimulation between the B6.ΔsNASP.lpr and B6.lpr groups, as well as the B6.ΔsNASP and B6.WT groups.Additionally, type I interferons (IFN-α) are key cytokines involved in the innate immune response and are implicated in the pathogenesis of autoimmune diseases, such as SLE, mediated by the IRF3 pathway [20,21].IFN-α also did not exert influence in B6.lpr mice (Figure S1).Thus, different LPS stimulation did not significantly affect the sensitivity of the TLR4-TBK1 signaling pathway following sNASP gene mutation, suggesting that the activation of TLR4 by LPS does not drive the IRF3 signaling pathway.In conclusion, these results suggest that the mutation in sNASP may induce a proinflammatory response and enhance the activation of TLR4 through TAK1 pathway (Figure 7).

Discussion
The female NZB/WF1, MRL/lpr, and BXSB/Yaa mice are the most commonly used animal models for SLE due to their natural development of immunopathological symptoms that resemble those observed in SLE patients [22].Obviously, lupus mouse model and SLE patients may have similar pathogenesis and some common genetic basis [23].Therefore, finding and identifying the pathogenic genes of lupus mouse model will greatly help to clarify the etiology and pathogenesis of SLE.NZW mice were backcrossed with NZB/WF1 mice repeatedly to obtain an inbred strain of NZM2410 mice.Both male and female mice could spontaneously develop the same autoimmune diseases as lupus NZB/WF1 mice.In 1994, it was first reported that three lupus susceptibility loci associated with autoimmune diseases are called Sle1, Sle2, and Sle3, respectively.The Sle2 locus is located on chromosome 4, which has been found to regulate B-cell hyperactivity and expansion of B1a cells in NZM2410 mice that some gene mutations from the Sle2 locus, could promote the inflammatory response, such as Skint6 and sNASP [6,[24][25][26].The variant of Skint6 W168X allele was confirmed as a pathogenic mutant gene that triggers autoimmune disease by producing a truncated Skint6 peptide binding the Skint6 receptors on T and B lymphocytes [26].
In addition, the variant of sNASP combined with the lpr mutation in the Fas gene enhances autoimmunity, resulting in more severe lupus nephritis and significantly increased lymphadenopathy in the B6.lpr strain [27].Traditionally, sNASP is crucial in assembling chromosomes and is vital in the final stages of DNA replication and folding chromosomes.It binds to H1 and H4, participates in histone transport, and promotes cell proliferation [28,29].sNASP also maintains histone H3K9me1 to regulate chromatin accessibility [6].Recently, another new biological function of protein sNASP has been discovered [10].The cytoplasmic protein sNASP can bind to tumor necrosis factor receptor-associated factor 6 (TRAF6) and prevent the ubiquitination of TRAF6 and its downstream signal pathway and the production of inflammatory cytokines in unstimulated macrophages.However, after TLR4 was stimulated by LPS, sNASP was phosphorylated and separated from TRAF6.Then, TRAF6, which does not bind to sNASP protein, produces autologous ubiquitin, activating downstream signal pathways and initiating the production of inflammatory cytokines.In addition to TLR4, the stimulation of TLR1, TLR2, TLR5, and TLR6 can also mediate the phosphorylation of sNASP and its dissociation from TRAF6, so the protein sNASP can also negatively regulate these TLR signaling pathways and the production of inflammatory cytokines.TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed on mononuclear macrophages, dendritic cells, and B cells, as well as on CD4 + T and CD8 + T cells [22].It is suggested that the negative regulation of protein sNASP on TLR signaling pathway exists in innate immunity and adaptive immunity-related immune cells.Aberrant activation of TLRs may disrupt immune homeostasis, which leads to excessive inflammatory cytokines aggravating the immune responses [30].Although at present, the relationship between TLR and SLE is mostly focused on TLR7 and TLR9 because they are in the cytoplasm and can recognize nucleic acid antigens.Until recently, accumulating evidence shows that TLR4 also plays an important role in the pathogenesis of SLE [11,31].A penetration-conjugated small peptide (TIP3) could block cytokine production to ameliorate inflammatory response in mice models of arthritis and alleviate the disease symptoms of SLE models through the TLR4 pathway [32].To test whether the mutation of sNASP gene alters the sensitivity of the TLR4 receptor, resulting in the difference between sNASP mutation groups and control groups in the activation level of TLR4 signaling, we measured the protein levels of the TLR4 signaling pathways and the quantified pro-inflammatory cytokine activations following different LPS stimulation in the peritoneal macrophages.As hypothesized, the mutation of sNASP produced excessive pro-inflammatory cytokines, specifically IL-6 and TNF-α at both mRNA and protein levels, which enhanced the activation of the TLR4-TAK1 signaling pathways.
Cytokine dysregulation aggravates immune dysfunction, leading to autoimmune diseases such as SLE [33].The proinflammatory cytokines, which infer IL-6 as a mediator of disease initiation, increase the susceptivity of the vascular pathology in MRL-Fas lpr mouse model [34].A meta-analysis suggested that the levels of IL-6 and TNF-α in SLE patients were higher than in healthy controls [35].Compared to B6. lpr, the proteins of IL-6 and TNF-α in macrophage culture supernatant in B6.ΔsNASP.lprmice were significantly higher, and the level of the mRNA was also increased by LPS stimulation.The same results were observed in B6.ΔsNASP and B6.WT mice, suggesting that the mutation in sNASP gene increases the expression levels of IL-6 and TNF-α genes and proteins in mouse peritoneal macrophages.To investigate how the mechanism increases the transcription of the proinflammatory cytokines, we measured the protein in TLR4 signaling pathways following the different stimulation of LPS.TLR4 is associated with two parallel downstream signaling pathways.The first pathway, known as the TLR4-TBK1 pathway, is MyD88-independent and involves the molecules TRAF3 and TBK1 [17,36].The second pathway, called the TLR4-TAK1 pathway, is MyD88-dependent and operates through the molecules TRAF6 and TAK1.These two pathways play crucial roles in transmitting signals downstream of TLR4 activation.Our results have shown that after stimulation of mouse peritoneal macrophages for 0.5, 1, and 2 hr by 1 ng/ml LPS, the levels of p-TAK1 and p-p38 in peritoneal macrophages of B6.ΔsNASP.lprmice were higher than those of B6.lpr mice only after stimulation for 2 hr.In the 10 ng/ml and 100 ng/ml LPS stimulation groups, the levels of p-TAK1, p-p65, and p-p38 in peritoneal macrophages of B6.ΔNASP.lpr mice were significantly higher than those of B6.lpr mice at 0.5 hr after LPS stimulation.Additionally, the expression levels of p-TAK1, p-p65, p-JNK, and p-p38 protein in peritoneal macrophages of B6.ΔNASP mice were higher than those of B6.WT mice after stimulated with 1 ng/ml LPS for 2 hr.The levels of p-TAK1, p-p65, p-JNK, and p-p38 in peritoneal macrophages of B6.ΔNASP mice were significantly higher than those of B6.WT mice at 0.5 hr after stimulation with 10 and 100 ng/ml LPS.These results indicated that the mutation in sNASP gene enhances the sensitivity of the TLR4 receptor in mouse peritoneal macrophages by the TLR4-TAK1 signaling pathway.Furthermore, compared with B6.ΔsNASP.lpr,B6.WT and B6.ΔsNASP mouse peritoneal macrophages, the   This study suggested that mutation of sNASP gene has no significant effect on TLR4-TBK1 signaling pathway in mouse peritoneal macrophages.

Conclusion
A variant of sNASP enhances the sensitivity of the TLR4 to aggravate the signaling pathway and increases the release of inflammatory cytokines.Despite the current knowledge of sNASP, further investigations are needed to elucidate whether the mutant sNASP exerts similar effects on other TLRs and whether its involvement in histone acetylation modification and DNA methylation, which may contribute to the SLE progression [24,37].These additional investigations will provide further insights into the broader impact of the mutant sNASP and its role in modulating immune responses associated with SLE.