Splenic ultrasound stimulation reduces arthritis severity

We used the K/BxN serum-transferred model of inflammatory arthritis. This model allows consistent induction of distal, symmetric polyarthritis when serum from T cell receptor transgenic, spontaneously arthritic K/BxN animals is injected intraperitoneally into recipient mice. Like rheumatoid arthritis in humans, the K/BxN serum-transferred arthritis causes inflammation due to IgG autoantibodies present in the serum14. Evaluation of the model in mice not exposed to US revealed considerable swelling in the ankles and paws in the days following serum transfer before naturally declining as the transferred antibodies were catabolized (Supplementary Fig. 1). Ankle thickness changes were measured with a caliper, and composite clinical scores were determined based on the established method of assessing rodent arthritis severity on a 0–12 scale; in brief, each paw was assigned a clinical score between 0 and 3, with 0 indicating no swelling as described in Methods15,16. Change in bodyweight was also monitored over the course of the study.

To investigate the effect of US treatment, mice were briefly anesthetized and treated noninvasively with spleen-targeted US or sham-US (control; same setup as treatment group except no energy delivered through the US transducer). We characterized the US pressure beam profiles, murine spleen dimensions, and spleen location to target the spleen using specific anatomical landmarks (Supplementary Fig. 2, 3 and Supplementary Table 1). Beginning one day prior to serum transfer, we administered daily US for 7 consecutive days (Fig. 1b), since the clinical score and ankle thickness tended to peak between day 7 and 10 (Supplementary Fig. 1). Each animal was monitored daily to document arthritis severity, and blood was collected on the final day of the experiment to validate successful transfer of arthritogenic antibodies (see Fig. 1b and Supplementary Methods).

Daily splenic US treatment with a 1 MHz transducer presented at 350 kPa for 2 min per day reduced the ankle thickness and clinical score by the conclusion of the 7-day experiment (Fig. 2a–d). We used 1 s on/5 s off US pulse pattern for our initial studies, because this was shown to have anti-inflammatory effects by Gigliotti et al.11,12. Several different pressures were evaluated, but 350 kPa demonstrated consistent therapeutic effects (see next section). Specifically, compared to sham-treated animals, US treatment significantly improved final day ankle swelling (p = 0.0017) and clinical scores (p = 0.0060) using a Mann–Whitney test. The progression of arthritis development and the effects of US treatment over a 7-day period can be observed in Fig. 3a, b, in which US stimulation gradually improved ankle swelling and clinical scores over time, which was consistently observed across multiple experiments (Supplementary Fig. 4). In order to objectively compare results across separate experiments that employed different US stimulation parameters and arthritogenic serum batches, the ankle swelling and clinical scores were normalized (Fig. 2c, d; see Methods). A normalized representation of the data from Fig. 2a, b is shown in Fig. 2c, d with positive values indicating a worsening of arthritis and negative values representing an improvement of arthritis. We further used this normalization method to optimize US treatment by comparing the therapeutic effects across different US parameters (carrier frequency, pressure amplitude, focal depth, and duration) and conditions (knock-out mice lacking T or B cells and treatment initiated before or after onset of arthritis) described below.

Fig. 2 US treatment modulates arthritis severity. Results from 7-day arthritis experiments are presented. a–d Pooled data from three experiments (total n = 32 mice), using identical experimental conditions (1 MHz US at 350 kPa, 1 s on/5 s off bursts for 2 min per day; shallow US-focusing cone to target the spleen, see Supplementary Fig. 2a) are shown. On the final day of the experiment, change in ankle thickness (a) and clinical score (b) of US treated animals are significantly reduced compared to sham-US controls (p = 0.0017 and p = 0.0060, respectively, using a Mann–Whitney Test). For comparison, normalized data from the same set of experiments are shown in c, d. A normalized value of zero indicates that the animal’s arthritis outcome was similar to the average untreated (sham-US) animal, a positive value indicates clinical worsening, and a negative value indicates clinical improvement. e, f Dose-response curves of US pressure reveal an optimal US amplitude between 333–350 kPa. Each point represents the normalized mean change of US treatment compared to sham at day 7. 1 MHz US stimulation with 1 s on/5 s off bursts for 2 min per day was applied in each experiment. Circles indicate that the shallow US-focusing cone was used, and triangles indicate that the deep US-focusing cone was used. Results are pooled from 108 mice across 9 experiments (see Mice section of Methods). g Additional US stimulation parameters and body locations were also tested, revealing consistent and effective therapeutic effects when specifically using 350 kPa, 1 s on/5 s off bursts with the shallow US-focusing cone targeting the spleen. One hundred and three mice were used over 9 experiments. All conditions consisted of 2 min of US stimulation per day, except for the right leg conditions that consisted of 4 min per day. Contra-spleen refers to right abdominal stimulation contralateral to the spleen. Tcra KO and muMt- were spleen stimulation experiments performed in T cell and B cell knockout mice, respectively. Mean and SEM are shown for all figures. Double asterisks denote p < 0.01. kPa kilopascal Full size image

Fig. 3 US treatment is effective before or after arthritis onset and depends on duration of stimulation period (page 18). All experiments in this figure used the 1 MHz US at 350 kPa, 1 s on/5 s off bursts in animals with arthritic serum transfer at day 0. a, b Full 7-day time course pooling data from the three experiments shown previously (Fig. 2a, b), which used identical experimental conditions for 2 min per day with US administered daily from day -1 through 6 (total n = 32). c, d Ultrasound duration causes a dose-dependent effect on arthritis. 6, 12, and 20 minute US stimulation was tested and is shown with the 2 min US and sham results from a, b for comparison. Figures show the 7-day progression of the disease and mice were stimulated with US from day -1 through day 6. For the 6, 12, and 20 min experiments, there were 6 animals per cohort (total n = 18) and for the 2 minute US and control data there were 16 animals per cohort (total n = 32). e, f US treatment is also effective at reducing symptoms after onset of arthritis. Treatment was not initiated until day 3 after arthritis had manifested. US was administered for 20 min per day. Data was pooled from two experiments with a total n = 23. g, h Normalized 7th day values of ankle thickness (g) and clinical score (h) for the different durations of ultrasound treatment shown in 3a–f. Error bars are SEM. 6, 12, and 20 minute values were normalized to the pooled sham control data from a, b. The bar “20 min US initiated on day 3” shows the improvement in ankle thickness and clinical score compared to the first day of treatment on day 3. In a–f, whisker plots are presented for better visualization of the data over time in which the endpoints of the vertical lines span the minimum to maximum values, the midline of each box is the median value and each box extends from the 25th to 75th percentile. In g, h, mean and SEM are plotted for each condition. kPa kilopascal Full size image

Identifying the optimum ultrasound pressure dose

We assessed whether the effect of US on arthritis severity depended on the pressure amplitude of stimulation by evaluating US pressures ranging from 25–1000 kPa, all delivered by a 1 MHz US transducer at 1 s on/5 s off bursts for 2 minutes per day. The normalized dose-response curves for US pressure amplitude and effect on arthritis severity are shown in Fig. 2e, f. These results reveal a U-shaped pressure dose response curve with an optimal US pressure of between 333–350 kPa, and suggest that arthritis-ameliorating effects depend on specific US pressure amplitudes.

Identifying therapeutic parameters and targeting

We investigated additional US parameters including acoustic carrier frequency, pulse pattern and focal depth, as well as target location. Stimulation of the spleen at an acoustic frequency of 220 kHz was not as effective at reducing arthritis as stimulation at 1 MHz, and the 1 s on/5 s off pulse pattern (16.7% duty cycle) was consistently more effective than the 2 ms on/8 ms off (20% duty cycle) pulse pattern when using 1 MHz (Fig. 2g). Custom-built US-focusing cones with different beam focal depths were compared (Supplementary Fig. 2). The shallow focusing cone was more effective at reducing the symptoms of arthritis (Fig. 2g), and was therefore used for a majority of the studies. We identified an effective treatment parameter setting using the 1 MHz frequency at 350 kPa with a 1 s on/5 s off pulse pattern while targeting the spleen with the shallow-focusing cone. We performed three separate experiments using this parameter and observed consistent therapeutic effects (Fig. 3a, b and Supplementary Fig. 4). All data points from those three experiments are pooled together and plotted in Fig. 2g in the left−most position (same data as in Fig. 2c, d). Stimulating other body areas such as the anterior and posterior thigh regions (Fig. 2g, Right Leg) or on the contralateral abdomen opposite to the spleen (Fig. 2g, Contra-Spleen) did not effectively reduce arthritis severity.

Daily US stimulation duration had a dose-dependent effect on arthritis (Fig. 3c, d). We tested 6-, 12-, and 20-minute US durations using the same parameters as before with a 1 MHz stimulus calibrated to 350 kPa and using a 16.7% duty cycle. Animals from the 6-, 12-, and 20-minute cohorts were handled identically and underwent the same dose of anesthesia per day (20 min of 1.5% isoflurane). Longer durations of treatment led to greater reductions in arthritis severity. Normalized data presented in Fig. 3g, h display this inverse relationship; notably, half of the animals receiving 20 min of daily US treatment exhibited almost no ankle swelling (values approaching −1). This duration dose-dependent trend is statistically significant for ankle thickness and clinical score, as shown in the Supplementary Fig. 5, which plots the linear regression of the response curve (p < 0.0001, R2 = 0.3839 and p = 0.0460, R2 = 0.1187 for ankle thickness and clinical score, respectively). Longer duration of US stimulation was also significantly correlated with reduced disease-driven weight loss (Supplementary Fig. 6).

Therapeutic effect of ultrasound depends on lymphocytes

As described in the Introduction, previous studies have proposed that splenic T and B cells contribute to the anti-inflammatory effects induced by electrical stimulation of the vagus nerve or by US stimulation of the spleen1,2,3,4,5,6,7,8,9,10,11,12. To investigate if T and B cells contribute to the arthritis-ameliorating effects of splenic US stimulation in our experiments, we performed two additional experiments in mice genetically lacking T or B cells. Splenic US did not consistently reduce arthritis severity in T cell or B cell deficient mice (Fig. 2g, “Tcra KO” and “muMt−”, respectively), suggesting that the anti-inflammatory effect of US in our model similarly depends on lymphocytes, either directly or indirectly. Interestingly, a few animals in both experiments did exhibit some improvement in inflammation, suggesting that the therapeutic mechanism may involve additional cell types.

Ultrasound can be used to prevent or treat arthritis

The results presented thus far involve initiation of ultrasound prior to induction of arthritis. We next tested whether US delivered after the onset of arthritis could still produce therapeutic effects. This is directly relevant for clinical applications since patients seek medical treatment after the onset of arthritis. All animals were injected with arthritogenic serum on day 0 but were otherwise undisturbed to allow arthritis to progress until day 3. On the third day, US or sham-US was initiated. We observed improvements in disease severity up to day 7, in which ankle thickness in the US cohort was significantly improved (p = 0.0490) and clinical score also trended toward improvement (p = 0.0900) using a Mann–Whitney test, as shown in Fig. 3e, f. The treatment was consistent across two independent experiments (total n = 23; Supplementary Fig. 7). For comparison with the results for the other duration parameters, these data are normalized to the average sham data from the same experiment and plotted in Fig. 3g, h. These experiments demonstrate that US stimulation targeting the spleen after onset of arthritis is also capable of improving the progression of the disease.

Single-cell RNA sequencing in ultrasound treated animals

We have demonstrated that the efficacy of US depends on specific targeting of the spleen, and that efficacy is reduced in animals lacking T or B cells. We therefore sought to determine the molecular mechanisms underlying the reduced joint inflammation seen with US treatment by investigating the gene expression profiles of lymphocytes in the spleen after 7 days of US therapy using single cell RNA sequencing (scRNA-seq). Spleen-targeted US or sham-US was delivered by a 1 MHz US transducer at 1 s on/5 s off bursts for 12 min per day, which was a clinically effective stimulation duration in our previous experiments and still enabled us to evaluate 16 mice within a reasonable time period per day. We compared four treatment groups: arthritogenic serum-injected mice that received sham-US or US treatment and control non-arthritogenic serum-injected mice that received sham-US or US treatment, with four animals in each group (n = 16, Supplementary Fig. 8b). For this analysis, CD45 + splenic leukocytes from the four mice in each treatment group were combined, and ~25,000 cells per treatment group were analyzed by scRNA-seq. Cells were filtered, normalized, and analyzed for heterogeneity in transcriptional profiles, and cell types were determined and assigned based on well-established cell-type markers. Here, we analyzed T cells or B cells for each treatment group. Cells were assigned by unbiased clustering based on a principal component analysis and expression of Cd3g (encoding CD3g) and Ms4a1 (encoding CD20) transcripts, which are known markers for T and B cells, respectively.

From this scRNA-seq data, we determined which genes were differentially expressed due to US stimulation. Among mice that received arthritogenic serum, we analyzed changes in gene expression in T and B cells that could be attributed to US treatment. The differentially expressed genes (DEGs) are shown in Fig. 4. We similarly identified DEGs in non-arthritogenic serum-injected mice (subjected to US or sham-US treatment; Supplementary Fig. 8a). In T cells from arthritic mice, we identified 8 DEGs with adjusted p-values <0.05 (ranging from p = 3.95e-230 to 5.04e-45 for the first seven genes, and p = 0.0239 for Iglc1 using a Wilcox rank sum test with Bonferroni correction) when comparing US-treated versus sham-US mice (Fig. 4 and Supplementary Fig. 8b). Similarly, we found 12 DEGs (US versus sham-US) in B cells in arthritic mice (adjusted p-values ranging from 9.76e-179 to 1.75e-34 using a Wilcox rank sum test with Bonferroni correction), and all but one were upregulated with US treatment (Fig. 4). Adjusted p-values show robust gene expression differences; however, only modest average log fold changes (logFCs) existed between the two treatment groups (Supplementary Table 2). These modest logFCs could result from a low percentage of cells expressing a particular gene or could indicate that the ultrasound treatment induces a relatively minor perturbation to the immune system. Interestingly, most of the DEGs found in arthritic mice were not differentially expressed in the non-arthritic mice (except mt-Atp8 and Iglc1 denoted with x in Fig. 4, Supplementary Fig. 8), suggesting a unique effect of US stimulation on arthritic mice. Additionally, 6 out of the 8 DEGs (bolded genes in Fig. 4) were upregulated in both T and B cells in arthritic mice, suggesting US induces overlapping effects in these two cell types. These findings demonstrate that US stimulation targeting the spleen induces significant changes in the transcriptional profiles of lymphocytes, and interestingly these changes are unique to the arthritic disease state. Our finding that genes in T and B cells are significantly differentially expressed with US treatment is consistent with literature showing these cell types to be involved in the splenic anti-inflammatory pathways4,7,8,–9.