Abstract
Pharmacological stimulation of the vagus nerve has been shown to suppress inflammation and reduce blood pressure in a murine model of systemic lupus erythematosus (SLE) that is characterized by hypertension, inflammation, renal injury and dysautonomia. The present study aims to directly stimulate vagal nerves at the level of the dorsal motor nucleus of the vagus (DMV) using designer receptors exclusively activated by designer drugs (DREADDs) to determine if there is similar protection and confirm mechanism. Female NZBWF1/J (SLE) mice and NZW/LacJ mice (controls, labeled as NZW throughout) received bilateral microinjections of pAAV-hSyn-hM3D(Gq)-mCherry or control virus into the DMV at 31 weeks of age. After two weeks of recovery and viral transfection, the DREADD agonist clozapine-N-oxide (CNO; 3 mg/kg) was injected subcutaneously for an additional 14 days. At 35 weeks, mean arterial pressure (MAP; mmHg) was increased in SLE mice compared to NZW mice, but selective activation of DMV neurons did not significantly alter MAP in either group. SLE mice had higher indices of renal injury including albumin excretion rate (μg/day), glomerulosclerosis index, interstitial fibrosis, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) compared to NZW mice. Selective DMV neuronal activation reduced albumin excretion rate, glomerulosclerosis, interstitial fibrosis, and NGAL in SLE mice but not NZW mice. Together, these data indicate that selective activation of neurons within the DMV by DREADD protects the kidney suggesting an important role of vagus-mediated pathways in the progression of renal injury in SLE.
Keywords: DREADD, Kidney injury, SLE, Vagal nerves, Cholinergic anti-inflammatory pathway, Inflammation, Glomerulosclerosis
1. Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disorder associated with abnormal immune responses, chronic inflammation and organ damage (Kiriakidou and Ching, 2020; Gergianaki et al., 2018). SLE in humans and animal models like the NZBWF1 mouse is characterized by autoantibody production, hypertension, renal inflammation, renal injury, and dysautonomia, i.e., decreased in vagal tone (Mathis, 2015a; Fairley and Mathis, 2017). Our previous findings indicate that the decrease in vagal tone impairs the cholinergic anti-inflammatory pathway (CAP), an endogenous vagus-to-spleen mechanism, and contributes to chronic inflammation in SLE mice (Pham et al., 2018a).
The neural circuit involved in CAP modulation involves the activation of brain areas such as the nucleus of the tractus solitarius (NTS), nucleus ambiguous (NA) and the dorsal motor vagus nucleus (DMV) (Pavlov et al., 2003). It has been shown that DMV lesion increases injury and inflammation and abolishes the protective effects of CAP stimulation by acetylcholine in an esophagitis rat model (Zhao et al., 2019). Furthermore, previous studies show that DMV neuron stimulation can increase vagus nerve activity and increase splenic nerve activity ultimately activating CAP (Booth et al., 2021).
Our lab already demonstrated that the pharmacological potentiation of efferent vagus nerve by systemic galantamine suppress inflammation and reduces blood pressure in SLE. However, although galantamine’s capability of crossing the blood-brain barrier, it is uncertain where galantamine is acting to increase CAP—if within the brain or peripherally (Pavlov et al., 2009; Pohanka, 2014). The present study aims to specifically stimulate vagal nerves within the DMV to directly enhance the CAP using designer receptors exclusively activated by designer drugs (DREADDs), a chemogenetic tool that is commonly used to target neurons in the CNS (Mukerjee and Lazartigues, 2019; Nation et al., 2016). Based on the aforementioned, we hypothesize that increasing vagus nerve activity through selective activation of neurons within the DMV will improve the disease outcome by decreasing disease severity and improving blood pressure, splenic and renal inflammation and renal injury due to the CAP activation in SLE mice.
2. Methods
2.1. Animal model
Female NZBWF1 mice, the first generation cross between New Zealand Black and New Zealand White (NZW), is an established model of SLE used in this study, with female NZW/LacJ mice serving as the controls. SLE and NZW mice were purchased at 3–6 weeks of age from Jackson Laboratories (Bar Harbor, ME) and aged/maintained on a 12-hour light/dark cycle in temperature-controlled rooms with food and water ad libitum. This study protocol was approved by the University of North Texas Health Science Center Institutional Animal Care and Use Committee (IACUC) and was in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.
2.2. Microinjections
To selectively target DMV neurons, DREADD (pAAV-hSyn-hM3D (Gq)-mCherry) or control (pAAV-hSyn-mCherry) virus (Control) was microinjected bilaterally at the DMV. Both SLE and NZW mice were anesthetized with isoflurane and placed in a stereotaxic frame. The following coordinates were used to access DMV with calamus as reference: 0 mm caudal, 0.25 mm lateral, and 0.48 mm ventral. Bilateral microinjections of 50 nL volume on each side were made using a pneumatic pico pump (World Precision Instruments, Sarasota, FL). To confirm the site of microinjection, the brain was harvested and incubated with 4 % PFA overnight and then in 30 % sucrose in PBS and cut using a cryostat. Slices that contain sections of DMV were mounted on slides and cover-slipped with Permount for microinjection verification.
2.3. Experimental design
Mice were separated into four experimental groups for the chronic experiment: 1) NZW/Control virus, 2) NZW/Gq DREADD, 3) SLE/Control virus, and 4) SLE/Gq DREADD. At 31 weeks of age, SLE and NZW mice received either DREADD or control virus at the DMV and were allowed to recover for 2 weeks. At 33 weeks, DREADD agonist, clozapine N-oxide (CNO; diluted in DMSO followed by 0.9 % saline (3 mg/kg), Tocris Bioscience, Minneapolis, MN), was administrated subcutaneously for 14 consecutive days to selectively activate DMV neurons. At 35 weeks of age, mice were housed in a metabolic cage for urine collection. Then, anesthetized mice were implanted with carotid catheters and allowed to recover. Blood pressure was recorded in conscious mice the following two days for 1.5 h each day as previously described (Morales et al., 2021). At the end of the study, mice were anesthetized with isoflurane and blood was collected, and then perfused with heparizined saline, and kidneys and spleen harvested and stored at −80 °C.
2.4. Index of disease severity
Blood samples were centrifuged for 20 min at 4 °C at 11,000 rpm for plasma collection. Anti-double-stranded autoantibodies (dsDNA), a hallmark of human SLE, were quantified using commercial ELISA kits (Alpha Diagnostic, San Antonio, TX) as previously described (Pham et al., 2020).
2.5. Western blotting
Spleen and kidneys were isolated in RIPA buffer containing protease Inhibitor (cOmplete™ Mini, EDTA-free Protease Inhibitor Cocktail -Roche®). BCA Protein Assay Kit was used to total protein quantification (Thermo Scientific™) so 100 μg of proteins were loaded equally and separated by electrophoresis followed by the transfer in PVDF membranes using Trans-Blot Turbo Transfer System - Bio-Rad. Then, membranes were blocked with blocking solution for 1 h and incubated overnight with primary antibody for tumor necrosis factor alpha (TNFα; Santa Cruz #52746). After 1-hour incubation a secondary antibody signal was measured by chemiluminescent with Clarity western ECL substrate mixture (Bio-Rad) (Pham et al., 2020). Volume intensity of TNFα protein band was normalized by total lane protein, determined by the stain-free total protein method expression using Image Lab software version 5.1 (Gilda and Gomes, 2013).
2.6. Renal injury determination
Urine collected at 35 weeks of age was used to quantify urinary albumin, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1). Urinary albumin was measured at 35 weeks of age via a commercial ELISA kit (Alpha Diagnostic). Albumin concentrations were divided by the total urine volume rate to get the albumin excretion rate (AER) presented as μg/day. To determine the extent of renal tubular injury, neutrophil gelatinase-associated lipocalin (NGAL; Cat# MLCN20; R&D Systems; Minneapolis, MN) and kidney injury molecule-1 (KIM-1; Cat# MKM100; R&D Systems; Minneapolis, MN) were determined by ELISA according to the manufacturer’s protocol.
2.7. Kidney histology
Left kidney was fixed in formalin, embedded in paraffin, then sliced at 6 μm. To verify glomerulosclerosis index, slices were stained with Periodic-Acid Schiff and 30 glomeruli from each subject were scored as previously described (Mathis et al., 2014). To quantify interstitial fibrosis, slices were stained with Masson’s trichrome staining kit (Catalog # KTMTR2; Stat Lab, McKinney, TX) according to the manufacturer’s protocol. Interstitial fibrosis was quantified by the percentage of the blue staining using NIS-Elements BR 4.30.01 software and same thresholding parameters were used for all the images to measure renal fibrosis. Images were captured using a Nikon Eclipse Ni microscope equipped with a Nikon DS-Fi2 color camera (Nikon) and NIS-Elements BR 4.30.01 software.
2.8. Acute activation of DMV neurons
A non-diseased mouse was anesthetized with isoflurane 0.25–5 % in 100 % oxygen and placed in a stereotaxic frame. The DREADD adeno-associated virus (pAAV-hSyn-hM3D(Gq)-mCherry; Addgene, 50 nL over 5 min) was bilaterally injected into the DMV and mice were allowed to recovery for 2 weeks. To confirm the activation of vagus nerve, a Millar® catheter (model SPR 671) was implanted into the carotid artery for heart rate recording in an anesthetized female NZW mouse given α-chloralose (80 mg/kg) and urethane (1000 mg/kg) as previously described (Pham et al., 2020). A midline cervical incision was made, and the right vagus nerve was localized, dissected from the connective tissue, and placed in a silver bipolar electrode embedded in a silicone-based elastomer. HR and vagus nerve activity was recorded for 5 min for baseline measurements and then DREADD agonist, clozapine-N-oxide (CNO) was injected subcutaneously (5 mg/kg) to selectively activate neurons within the DMV. Response was recorded for 10 min and brain was collected for microinjection site confirmation.
2.9. Statistical analysis
Two-way ANOVA followed by Holm-Sidak post-hoc was performed to compare treatment groups using Prism - GraphPad. Statistical differences were considered when p ≤ 0.05 and statistical results are listed within the figures and figure legends.
3. Results
3.1. Targeted DMV neuron activation does not affect disease severity and MAP in female mice with SLE
Fig. 1 shows the ability of DREADD injection into the DMV to reduce heart rate (bpm) and increase vagus nerve activity once CNO is administered in acute experiments. In the chronic experiment, DMV neuron activation via DREAD did not change dsDNA in SLE (SLE/Control: 387264 ± 133,520 vs. SLE/Gq DREADD: 454335 ± 133,897, p = 0.9354) or NZW mice (NZW/Control: 114609 ± 53,397 vs. NZW/Gq DREADD: 242973 ± 103,974, p = 0.7502) (Fig. 2A). Mean arterial pressure (MAP) was increased in SLE mice compared to NZW control mice (149 ± 6 vs. 126 ± 4, p = 0.0076), but DMV neuron activation did not change MAP in either group (SLE/Gq DREADD: 140 ± 3 or NZW/Gq DREADD: 132 ± 2, p = 0.3455) as shown in Fig. 2B.
Fig. 1.
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Fig. 2.
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3.2. DMV neuronal activation did not change kidney and spleen inflammation in SLE
TNFα protein expression in spleen was increased in SLE compared to controls (1.9e6 ± 4.2e5 vs. 5.5e5 ± 8.8e4, p = 0.015) (Fig. 3A). DMV neuron activation did not change splenic TNFα expression in SLE mice (1.1e6 ± 1.3e5, p = 0.17) or controls (5.6e5 ± 1.2e5, p = 0.9782). There was no difference in TNFα protein expression in the kidney between any of the groups (NZW/Control: 1.8e6 ± 6.6e5, NZW/Gq DREADD: 2.2e6 ± 5.0e5, SLE/Control: 5.8e6 ± 1.6e5 and SLE/Gq DREADD: 5.6e6 ± 8.4e5) (Fig. 3B).
Fig. 3.
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3.3. DMV activation decreases renal injury in SLE female mice
Albumin excretion rate (μg/day), was increased in SLE mice compared to NZW control mice (1.3e4 ± 5.6e3 vs. 43 ± 11, p = 0.0676) and DMV stimulation with DREADD decreased albumin excretion rate in SLE mice although not significantly (225.8 ± 172.6, p = 0.0631) (Fig. 4A). SLE female mice had increased glomerulosclerosis index compared to control (2.836 ± 0.339 vs. 1.225 ± 0.0348, p = 0.0004) and DMV neuronal activation decreased glomerulosclerosis in SLE mice (1.670 ± 0.175, p = 0.0033), but not in controls mice (1.140 ± 0.015) (Fig. 4B). Interstitial fibrosis, verified by Masson’s trichrome staining, was increased in SLE mice compared to NZW mice (1.52 ± 0.46 vs. 9.61 ± 3.68, p = 0.0379) and efferent vagus nerve activation by DREADD decreased interstitial fibrosis in SLE (0.99 ± 0.34, p = 0.0379) and had no effect on NZW mice (0.57 ± 0.19, p = 0.9836) (Fig. 4C). Urinary NGAL was increased in SLE compared to NZW mice (2.7e5 ± 7.4e4 vs. 4.2e4 ± 1.9e4, p = 0.0564) (Fig. 4D). DMV activation decreased NGAL in SLE mice (2.9e4 ± 4.1e3, p = 0.0389) but not in NZW mice (3.2e4 ± 5.3e3, p = 9986). Kidney injury molecule-1 (KIM-1) was increased in SLE compared to NZW mice although not statistically different (1.6e4 ± 2.4e3 vs. 7.8e3 ± 1.2e3, p = 0.0662) and selective activation of DMV neurons decreased KIM-1 in SLE mice (7630 ± 1653, p = 0.0475) but not NZW mice (6.6e3 ± 1.3e3, p = 0.9813) (Fig. 4E).
Fig. 4.
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4. Discussion
In the current study, we tested the hypothesis that targeted activation of the vagus nerve at the level of the DMV would activate the efferent cholinergic anti-inflammatory, thereby reducing inflammation and preventing the development of hypertension and renal injury in female mice SLE. We found that DREADD and subsequent CNO administration increases efferent vagus nerve activity and significantly reduced renal injury in female mice with SLE. The effect of chemogenetic stimulation of the efferent vagus did not significantly alter splenic and renal inflammation or blood pressure.
SLE, a chronic autoimmune inflammatory disease that predominantly affects females, is characterized by a breach of immunological tolerance, leading to the production of overactive self-targeting T and B cells (Weindel et al., 2015; Zucchi et al., 2022). The presence of antinuclear antibodies and dsDNA autoantibodies are found in 70 % of patients and are known to correlate with SLE disease severity (Sabio et al., 2011). SLE is also associated with dysregulation of cytokine production, which contributes to immune dysfunction, tissue inflammation, and organ damage. Common major players in SLE are proinflammatory cytokines like type I and type II interferons (INFs), interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha (TNF-α) (Munguia-Realpozo et al., 2019). These increased inflammatory cytokines can promote chronic inflammation locally in the kidneys, leading to renal injury and dysfunction as well as hypertension (Ohl and Tenbrock, 2011).
Chronic kidney inflammation has been implicated in the development of hypertension (Rodriguez-Iturbe et al., 2012). The prevalence of hypertension reaches as high as 77 % in some cohorts in SLE patients, significantly higher than the roughly 8 % prevalence in non-SLE control patients (Bateman et al., 2012). Additionally, SLE patients are roughly five–six times more at risk of a cardiovascular event compared to the age-matched general population (Manzi et al., 1997). The exact underlying pathophysiologic mechanism for how kidney inflammation leads to hypertension is unclear, but glomerular damage and renal vascular endothelial dysfunction are likely contributors.
The NZBWF1 mouse is an established model for studying SLE as it spontaneously develops lupus-like symptoms around 20 weeks of age which closely mirror those of human SLE patients (Perry et al., 2011). Similar to human patients, female NZBWF1 mice are generally much more affected than males. We and others have demonstrated that these mice produce the same dsDNA autoantibodies characteristic in human SLE patients, and that there is a significant rise in dsDNA autoantibodies when these mice develop lupus symptoms (Lambert and Dixon, 1968). In this study, although dsDNA autoantibodies in SLE mice are greater than control mice, there was no interaction to enable comparison amongst the four groups. This is likely be due to the use of the NZW mice as the control group as they can also develop autoimmunity. The NZBWF1 mouse model also experiences an upregulation of proinflammatory cytokines, leading to the development of renal inflammation, renal injury, and hypertension (Ryan, 2009).
The cholinergic anti-inflammatory pathway was first described in 2003 by Wang et al. as a neuroimmune mechanism that regulates inflammation (Wang et al., 2003). This pathway is the efferent arm of the inflammatory reflex and is one of the endogenous compensatory mechanisms necessary to suppress inflammation (Mannon et al., 2019). The pathway begins at the efferent vagus nerve, the major parasympathetic nerve which innervates organs such as the heart, lungs, and digestive tract (Rosas-Ballina and Tracey, 2009). Stimulation of this nerve will subsequently activate the splenic nerve, resulting in the release of norepinephrine from nerve terminals. Norepinephrine then binds to its β2 receptor on the surface of specialized T cells (TChAT cells) to increase the release of acetylcholine, which is anti-inflammatory in the spleen. Acetylcholine can then bind to α7 nicotinic acetylcholine receptors (α7 nAChRs) on macrophages and other immune cells, inhibiting the production and release of cytokines such as TNF-α, IL-1, IL-8, and HMGB1 (Matteoli and Boeckxstaens, 2013).
It was already demonstrated that activation of the cholinergic anti-inflammatory pathway is beneficial in reducing inflammation in the angiotensin II-dependent model of hypertension (Wu et al., 2021), obesity (Wang et al., 2011), and diabetes (Chang et al., 2019). We have previously studied the cholinergic anti-inflammatory pathway using NZBWF1 mice and our published finding suggests that a decrease in vagal tone impairs the pathway, leading to chronic inflammation in the SLE model. Our hypothesis in the current study was that increasing vagus nerve activity through neuron activation at the dorsal motor vagus nucleus would activate the cholinergic anti-inflammatory pathway, ultimately leading to an improvement of SLE disease outcomes in NZBWF1 mice.
We found that increasing vagus nerve activity through activation of the dorsal motor vagus nucleus did not ultimately affect disease severity or MAP in female mice with SLE. We also found DREADD activation did not change inflammation in the kidneys or spleen, as TNF-α levels in both were unchanged due treatment. There may be several possible reasons for that. One reason may be different cytokines other than TNF-α are linked to changes in renal injury outcomes during the advanced disease. Age at which the mice received treatment (31 weeks) was too late, and that using DREADD to activate the DMV at an earlier age may be beneficial in preventing the onset of severe symptoms. NZBWF1 mice begin developing lupus symptoms at approximately 20 weeks of age. Future studies could administer DREADD before or shortly after this age point in order to slow down the pathogenesis of SLE.
We did find that increasing vagus nerve through activation of the DMV successfully decreased renal injury in female SLE mice. DREADD activation significantly decreased glomerulosclerosis, interstitial fibrosis, urinary NGAL, and KIM-1 in SLE mice while not affecting our control mouse model. Additionally, while not statistically significant, treatment did result in a trend towards a decrease in albumin excretion rate. Taken together, vagus nerve stimulation through the DMV had a clear beneficial and protective effect on kidneys of female SLE mice. One of the limitations of this study was the lack of outcomes to measure renal function. However, our findings opens pathways to explore the positive impact that activation of the CAP pathway has on kidney function in future studies.
Despite improving renal injury, targeted vagus nerve stimulation through the DMV was unable to alleviate SLE hypertension, potentially because renal and splenic inflammation were not affected by treatment. As mentioned above, earlier treatment may be necessary to have the desired protective effect against inflammation and, by extension, hypertension. Additionally, failure to alleviate renal inflammation following DREADD could describe a disconnect between vagal nerves originating from the DMV and the kidney. This brings attention to vagus nerves housed in other brain regions (i.e., NTS and NA) that could be crucial in this pathway. Finally, our data may suggest that the anti-inflammatory effect of vagus nerve stimulation in the spleen may not be directly linked to the anti-inflammation in the kidney as previously hypothesized (Mathis, 2015b; Pham et al., 2018b). Future work from our lab will elucidate the importance of the cholinergic anti-inflammatory pathway in the control of kidney inflammation.
Acknowledgments
We would like to thank Calvin D. Brooks, graduate student at the University of North Texas Health Science Center, for his technical assistance with experiments described within this manuscript. This study was funded by R01HL153703.
Data availability
Data will be made available on request.
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Associated Data
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Data Availability Statement
Data will be made available on request.
