Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease with multiple organ involvement. SLE is commonly diagnosed in women during their reproductive years (20–40 years), and, at least in the United States, the disease has a female-male ratio of 9:1 (Margery-Muir et al., 2017). The incidence of SLE varies between geographic locations and by racial or ethnic demographics, with people of black ethnicity having a much higher rate of incidence and prevalence than people of white ethnicity (Rees et al., 2017). Primary features of SLE include autoantibody generation against nuclear material, immune complex deposition in target tissues, including the joints, skin, and kidneys, leading to inflammation and organ damage (Moulton et al., 2017). Impairment in immune complex clearance also occurs and is a crucial pathological mechanism in SLE. Lupus nephritis constitutes one of the most concerning manifestations of SLE, occurring in approximately 30–60% of adult SLE patients and 50–80% of juvenile-onset patients (Pons-Estel et al., 2011; Massias et al., 2020). Up to 20% of lupus patients develop end-stage kidney disease within 10 years of diagnosis (Tektonidou et al., 2016), and a 2016 meta-analysis reported that SLE patients have over a 4-fold increased risk of mortality due to renal disease (Lee et al., 2016). Cardiovascular disease, including coronary artery and cerebrovascular disease, is also a major cause of morbidity and mortality among SLE patients (Burkard et al., 2018; Kostopoulou et al., 2020). Indeed, SLE patients carry at least a 2-fold increased risk of mortality due to cardiovascular disease (Lee et al., 2016), with estimates as high as a 50-fold increased risk of myocardial infarction compared to age- and sex-matched controls (Manzi et al., 1997). The risk of myocardial infarction and cardiovascular mortality have been shown to be even higher in lupus nephritis patients than in SLE patients without lupus nephritis (Hermansen et al., 2017). Part of this elevated risk may be attributable to the observation that SLE patients display premature onset and accelerated progression of atherosclerosis, although the underlying mechanisms are not fully understood (Kostopoulou et al., 2020). Nonetheless, several studies have reported that SLE patients display an elevated risk of cardiovascular events and mortality compared to people without SLE who have similar patterns of traditional cardiovascular risk factors (Goldberg et al., 2009; Ajeganova et al., 2021). SLE patients are often treated with corticosteroids due to their anti-inflammatory properties, but long-term use of these therapies carries a risk of adverse effects on metabolism. As the cardiovascular morbidity and mortality in SLE patients is not fully explained by traditional risk factors alone, this may indicate a synergistic effect of traditional risk factors and SLE, or treatments such as corticosteroids, to promote excess risk (Kostopoulou et al., 2020; Jha et al., 2022). As such, better understanding of cardiovascular disease in the context of SLE is vital to improving patient outcomes.

One of the major drivers of cardiovascular and renal risk in the general population is the “Metabolic Syndrome” (MetS). MetS describes a cluster of metabolic risk factors, including central obesity, insulin resistance, glucose intolerance, hypertriglyceridemia, and hypertension, which promotes the development of type 2 diabetes and cardiovascular disease (Bagheri et al., 2021). Recent meta-analyses report that MetS is approximately twice as common in patients with SLE than in healthy controls (Sun et al., 2017; Hallajzadeh et al., 2018). Highlighting the clinical impact of these comorbidities, studies conducted over the past 15 years in SLE patients from multiple regions around the world report that the presence of MetS is predictive of organ damage accrual as measured via the Systemic Lupus International Collaborating Clinics American College of Rheumatology Damage Index (SLICC/ACR DI) (Mok et al., 2018; Apostolopoulos et al., 2020; Ugarte-Gil et al., 2022). Together, these studies speak to a need for further investigation of how cardio-metabolic risk factors affect outcomes in SLE and whether SLE itself contributes to the development of MetS.

Animal models of SLE provide an opportunity to further study the interactions between SLE and MetS. Of the spontaneous mouse models of SLE, the female (New Zealand Black x New Zealand White) F1 mouse (NZBWF1) has been the most extensively characterized with respect to MetS phenotypes. Female NZBWF1 mice display hypertension, adiposity, hyperinsulinemia, and impaired glucose tolerance even on normal rodent chow (Ryan et al., 2006; Vilà et al., 2012). Feeding NZBWF1 mice a 45% kcal high-fat diet for 14 weeks accelerates the onset of albuminuria and worsens endothelial dysfunction (Gilbert and Ryan, 2011). A long-term high-fat diet has also been reported to induce development of atherosclerotic lesions in NZBWF1 mice (Hahn et al., 2010). Vasculitis, arterial lipid deposition and myocardial infarctions were also seen following feeding of an atherogenic diet to another spontaneous mouse model of lupus, the MRL/lpr mouse (Qiao et al., 1993). As such, these spontaneous mouse models of SLE appear vulnerable to the development of MetS and diet-induced cardiovascular complications, highlighting the translational relevance of the models.

Inducible models of SLE offer the opportunity to more easily probe the roles of specific genes by superimposing SLE onto a transgenic mouse of interest. A key pathway leveraged in multiple currently available inducible mouse models of SLE is toll-like receptor 7 (TLR7) activation. Enhanced signaling through toll-like receptors, including TLR7, has been implicated in promoting autoimmune disease (Farrugia and Baron, 2017). TLR7 is an X chromosome-encoded endosomal receptor that recognizes single-stranded RNA. Genome-wide association studies report polymorphisms in the TLR7 locus segregating with SLE (Shen et al., 2010; dos Santos et al., 2012) and more recently, a gain-of-function mutation in TLR7 was identified in a pediatric SLE patient (Brown et al., 2022). Proposed mechanisms by which TLR7 activation may contribute to SLE include through stimulating type I interferon production, expansion and plasma cell differentiation of autoreactive double-negative B cells, germinal center formation, and enhancing production of autoantibodies to RNA-containing antigens (Christensen et al., 2006; Subramanian et al., 2006; Lee et al., 2008; Jenks et al., 2018). As recently reviewed, an imbalance of the pathogenic role of TLR7 and protective role of TLR9 on effector function of B cells may also contribute to SLE (Fillatreau et al., 2021). The hydrocarbon oil pristane has been used to induce SLE in mice, working via a TLR7-dependent pathway (Lee et al., 2008). Whether the inducible pristane model of lupus is associated with features of MetS other than hypertension does not appear to have been explored. A more recently-developed inducible model of SLE is the treatment of mice with a TLR7 agonist such as imiquimod (Yokogawa et al., 2014). Taken together with the need to better understand the interaction between SLE and MetS, the purpose of the current study was to determine whether combined TLR7 agonist treatment and exposure to a high fat, high sucrose “Western diet” intervention affects the early-stage development of SLE or MetS in comparison to a high sucrose control diet.

Given the increased prevalence of MetS in SLE patients (Sun et al., 2017; Hallajzadeh et al., 2018) and the importance of MetS in promoting cardiovascular and renal disease, a better understanding of how these comorbidities interact is essential to improving the health of SLE patients. The goal of the present study was to test the early-stage effects of dual exposure of C57BL/6 mice to stimuli that promote the development of SLE and MetS. Based on the observation of elevated fasting blood glucose, hyperinsulinemia, adiposity, and development of splenomegaly, our data suggest that HFD plus IMQ treatment may provide such a model for further studying the synergistic interplay of the mechanisms underlying both conditions. Moreover, a striking finding of the current study was the apparent acceleration of the development of hyperinsulinemia in IMQ-treated HFD-fed mice. Previous studies of NZBWF1 mice revealed impaired glycemic control, hyperinsulinemia, adiposity, and hypertension even on normal rodent chow (Ryan et al., 2006; Vilà et al., 2012). Together, these findings suggest that factors associated with SLE or autoimmunity rather than iatrogenic effects of treatments may render patients more susceptible to developing MetS.

Whether TLR7 plays a role in MetS has not been extensively investigated, but a diffuse body of literature so far provides evidence that it might. TLR7 was among several differentially-upregulated genes identified in the livers of a nonalcoholic steatohepatitis model in minipigs that was induced by chronic feeding of a high-fat, high-sucrose diet (Xia et al., 2014). In a community-based observational study of the Framingham Heart Study Offspring Cohort, TLR7 mRNA levels in platelets isolated from women showed a small but statistically significant association with body mass index, total cholesterol to HDL ratio, and serum C-reactive protein (Koupenova et al., 2015). Conversely, male CD-1 mice fed a high-fat diet showed downregulation of TLR7 mRNA in liver and adipose tissue (Betanzos-Cabrera et al., 2012). TLR8 knockout mice have high endogenous TLR7 expression and activity that promotes a lupus-like phenotype (Demaria et al., 2010). The impaired glucose tolerance and hepatic inflammation that developed in TLR8 knockout mice after feeding a 60% kcal fat diet for 18 weeks was attenuated by double knockout of both TLR7 and 8 (Hanna Kazazian et al., 2019). TLR7 knockout mice also had improved glucose tolerance compared to wild-type control mice (Hanna Kazazian et al., 2019). As described above, NZBWF1 mice display hyperinsulinemia and impaired glucose tolerance (Ryan et al., 2006). The autoimmunity and glomerulonephritis observed in NZBWF1 mice are partially TLR7-mediated (Murakami et al., 2021), but whether the hyperinsulinemia and impaired glucose tolerance are also related to TLR7 activation has not been studied to our knowledge. Together with our data, these studies show potential for the involvement of TLR7 signaling in the development of dysglycemia and hyperinsulinemia, although the mechanisms underlying these effects remain to be further elucidated. A role for TLR7 in promoting inflammation of the liver (Hanna Kazazian et al., 2019) may be relevant to the disturbances of glycemic control in MetS and the high prevalence of dysglycemia and hyperinsulinemia SLE, given the liver’s central role in whole-body glucose homeostasis; further investigation in this area appears warranted.

Excessive body weight and adiposity are key components of the MetS and associated with the future development of insulin resistance (Hayashi et al., 2008; He Y. et al., 2020). Further, obesity appears to worsen autoimmune disorders in a number of incompletely understood ways, including promoting autoantibody production and secretion of adipokines such as leptin that promote inflammation (Tsigalou et al., 2020). The status of leptin in SLE has been somewhat controversial, but a 2020 meta-analysis (Yuan et al., 2020) reported overall elevated plasma leptin levels among female SLE patients, and some indication that a leptin receptor gene variant (rs1137101) may confer increased susceptibility to SLE. An increased frequency of leptin receptor-positive B cells in SLE patients and an effect of leptin to promote B cell cytokine production and differentiation to highly IgG and IgM-secreting plasma cells was also recently reported (Chen et al., 2022 Clin Exp Rheum). In the current study, the body weights and visceral (gonadal) fat deposition were found to be elevated in HFD-fed animals when compared with control animals, but IMQ-treatment mildly attenuated this difference. Similarly, plasma leptin concentrations were also increased by HFD but mildly attenuated within the same group. This apparent mild inhibitory effect of TLR7 activation on development of diet-induced obesity and hyperleptinemia contrasts with a mild protective effect of TLR7 knockout noted by Hanna Kazadian et al. (Hanna Kazazian et al., 2019). The reasons for this difference remain currently unclear, however the slightly but significantly reduced adiposity in IMQ-treated HFD mice does not seem to have been caused by a reduction of food intake. Nonetheless, HFD-feeding provides an environment of increased adiposity and hyperleptinemia, allowing further study of interactions between obesity and TLR7-mediated autoimmunity.

SLE patients have altered lipid profiles characterized by increased triglycerides and reduced HDL cholesterol concentrations (Chung et al., 2007). Although triglycerides were not measured in the current study due to insufficient plasma for analysis, we found increased total cholesterol levels among HFD-fed mice compared with control diet-fed mice. Interestingly, HDL rather than LDL/VLDL levels were elevated in the HFD-fed mice. Per the manufacturer’s specification sheet, the main fat source in the Teklad TD88137 HFD is anhydrous milk fat. An elevation of HDL cholesterol in C57BL/6 mice fed a high fat diet where the fat came predominantly from milk fat has been reported previously by others (Podrini et al., 2013). NZBWF1 mice exposed to a high fat “Western” diet also developed elevated HDL cholesterol, which included so-called “pro-inflammatory” HDL cholesterol that has pro-oxidant rather than antioxidant properties, as described by (Hahn et al., 2010). Whether the elevated HDL cholesterol measured in the HFD groups in the current study also included pro-inflammatory HDL cholesterol is unknown, as we had insufficient plasma available to determine this. Regardless and in contrast to effects on plasma insulin, IMQ treatment did not significantly affect any of the cholesterol parameters measured in the current study. He et al. (He M. Q. et al., 2020) found that LDL cholesterol and total cholesterol were more elevated after 9 weeks of a low sucrose, 60% fat diet protocol in C57BL/6 mice than after 5 weeks; therefore, a longer duration of study and perhaps a different diet composition to that used in the current study would be beneficial for studying the impact of TLR7-driven autoimmunity on other components of dyslipidemia.

Various dietary approaches have been used to model MetS in rodents, with chow containing high fat and high carbohydrate (often fructose) used singly or in combination, or drinking water containing sucrose or fructose (Wong et al., 2016). The high-fat, high-sucrose “Western” diet used in the current study (TD88137) was chosen rather than high fat alone in part to incorporate excess dietary sugar intake, which is common in the MetS population at large. Highlighting the importance of incorporating both high fat and high sucrose in understanding inflammatory outcomes, a recent study of IMQ-induced psoriasiform dermatitis noted an exaggerated inflammatory response to a similar high-fat, high-sucrose western diet to the one used in the current study than to a diet higher in fat alone (Yu et al., 2019). A caveat to the interpretation of differences between our control and HFD groups is, however, that the control diet used in the current study was a modified version of the TD88137, containing a matched sucrose level. A paired, low sucrose diet was not available. Selection of this control diet was done to allow the best possible consistency in other dietary components such as protein sources (casein). As such, the diet-induced differences between the groups in the current study are presumptively high fat-driven.

Splenomegaly is a common feature of SLE models (Du et al., 2015) and observed in both the IMQ-treated mouse groups in the current study, as well as by Yokogawa et al. (Yokogawa et al., 2014). Anti-dsDNA autoantibodies are common in SLE and typical for lupus nephritis, although autoantibodies also develop against other nuclear components. Indeed, the suite of autoantibodies developed (and organ involvement) differs in a strain- and genetic background-dependent manner in both spontaneous models such as the NZBWF1 as well as in inducible pristane and IMQ models (Reeves et al., 2009; Yokogawa et al., 2014). Yokogawa et al. (Yokogawa et al., 2014) reported an increase in anti-dsDNA autoantibodies in FVB/N and BALB/c mice after 4 weeks of IMQ treatment; data for C57BL/6 mice were not reported. We did not detect a significant rise in anti-dsDNA autoantibodies in female C57BL/6 mice in the present 6-weeks study. Similar to our study, treatment of male and female C57BL/6 mice with another TLR7 agonist, resiquimod, over a similar time-frame promoted splenomegaly, no significant rise in dsDNA autoantibodies but a significant increase in ANA (Wirth et al., 2020). Similarly to the absence of albuminuria in the C57BL/6 mice treated with IMQ in the current study, albuminuria had not developed after 5 weeks of resiquimod treatment of C57BL/6 mice (Wirth et al., 2020). Wirth and others (Wirth et al., 2020) did, however, note early mortality with 67% loss of female C57BL/6 mice by 6 weeks, but this is not something that we encountered in the current study or with longer treatment of transgenic mice on a C57BL/6 background in other ongoing work in our laboratory (unpublished data, not shown). The IMQ model is capable of producing glomerular Ig deposition and glomerulonephritis in other genetic backgrounds such as BALB/c and FVB/N (Yokogawa et al., 2014). Together, these data are consistent with TLR7 agonists promoting features of SLE in a strain-dependent manner, with additional variability within strains but between institutions possible for reasons that are as yet unclear.

Hypertension is an important risk factor for cardiovascular disease and progression of renal damage, and is prevalent in SLE patients. Female BALB/c mice treated with IMQ display elevated blood pressure as measured by tail-cuff plethysmography after 6 weeks (Robles-Vera et al., 2020). We did not measure arterial blood pressure in the present study and so cannot draw any conclusions regarding whether blood pressure was increased in any group. We did not, however, detect a significant effect of IMQ to induce left ventricular hypertrophy. The mild attenuation of the HFD-induced left ventricular hypertrophy in the IMQ-treated group is consistent with the overall attenuation of HFD effects on body weight and fat pad mass. The susceptibility of C57BL/6 mice to induction of hypertension by IMQ treatment remains to be determined.

The present study found that co-treatment of female C57BL/6 mice with HFD and IMQ induced dysglycemia, hyperinsulinemia, adiposity, and splenomegaly and ANA, indicating the induction of MetS and early stages of autoimmunity. This model may be useful for studying the role of TLR7 signaling in promoting or accelerating the development of dysglycemia and hyperinsulinemia in the context of diet-induced MetS.