key: cord-354765-abayh871
authors: Graham, R. S.; Zachs, D. P.; Cotero, V.; DAgostino, C.; Ntiloudi, D.; Kaiser, C. R.; Graf, J.; Wallace, K.; Coleman, T. R.; Ashe, J.; Pellerito, J.; Tracey, K. J.; Binstadt, B.; Chavan, S. S.; Zanos, S.; Puleo, C.; Peterson, E.; Lim, H. H.
title: Calming the Cytokine Storm - Splenic Ultrasound for Treating Inflammatory Disorders and Potentially COVID-19
date: 2020-07-17
journal: nan
DOI: 10.1101/2020.07.14.20153528
sha: 
doc_id: 354765
cord_uid: abayh871

Hyperinflammation and uncontrolled cytokine release, which can be seen in severe cases of COVID-19, require therapy to reduce the innate immune response without hindering necessary adaptive immune mechanisms. Here, we show results from the first in-human trials using non-invasive ultrasound stimulation of the spleen to reduce cytokine release in the context of both an acute response in healthy subjects and a chronic inflammatory condition in rheumatoid arthritis patients. Splenic ultrasound results in a reduction in TNF serum levels, as well as IL-1B; and IL-8 transcript levels in monocytes. There is also a down regulation of pathways involved in TNF and IL-6 production, and IFNgamma- and NFKB-regulated genes. Many of these cytokines or pathways are upregulated in COVID-19 patients. There is also a reduction in chemokine transcript levels and other components of the chemotactic response, suggesting that reduction of cellular migration may contribute to the therapeutic effects of ultrasound. There is no inhibition of the adaptive immune response with ultrasound treatment relating to antibody production. This is consistent with a pre-clinical animal model where enhanced antibody production was achieved with splenic ultrasound. Therefore, this new splenic ultrasound approach has the potential to treat acute and chronic hyper-inflammatory diseases, as it lowers cytokine levels without disrupting the normal adaptive immune response. Portable ultrasound technologies are currently being developed and translated to the clinic to treat various inflammatory disorders, with more recent efforts directed towards combatting the hyperinflammation or cytokine storm in COVID-19 patients.

Cytokines release by cells of the innate immune system drive inflammation (1, 2). Inflammatory reactions are typical in response to microbial or viral infection but can lead to health problems or life-threatening conditions if there is a persistent hyperactive innate immune response, involving cytokine toxicity and tissue damage (2, 3) . A recent and devastating example is the hyperinflammation caused by the coronavirus disease 2019 (COVID-19, disease associated with SARS-CoV-2 viral infection). It is estimated that 17% of COVID-19 cases experience a 'cytokine storm' that leads to severe or fatal respiratory disease, known as acute respiratory distress syndrome (ARDS) (4) (5) (6) . In addition to other acute conditions, such as sepsis and acute kidney injury (7, 8) , hyperinflammation is an issue that occurs across multiple chronic inflammatory systemic diseases, such as rheumatoid arthritis (RA) and irritable bowel syndrome (9, 10) . The current pharmacologic approaches to treating hyperinflammation are associated with multiple side effects and high costs.

Over the past 20 years, researchers within the field of bioelectronic medicine have investigated an unconventional approach for treating inflammation through the use of vagus nerve stimulation (11) (12) (13) . Electrical stimulation of the vagus nerve activates the splenic nerve and cells within the spleen, leading to activation of the cholinergic anti-inflammatory pathway (14, 15) . This pathway depends on splenic nerve release of norepinephrine that activates acetyltransferase -expressing T lymphocytes, which in turn modulates innate immune cells to decrease systemic levels of key proinflammatory cytokines, such as IL-6, IL-15 and TNF (16, 17) . Furthermore, electrical vagus nerve stimulation has been shown in mice and humans to provide therapeutic anti-inflammatory effects for chronic inflammatory diseases (e.g., RA and irritable bowel syndrome) and for acute inflammation (e.g., sepsis, renal ischemia, trauma/hemorrhagic shock, and acute lung injury following trauma/hemorrhagic shock; (16, (18) (19) (20) (21) .

Direct vagus nerve stimulation requires invasive implantation procedures. Several groups have pursued non-invasive ultrasound stimulation of the spleen as a safer, non-surgical approach for modulating the cholinergic anti-inflammatory pathway. Since the vagus nerve projects to the brain and multiple organs throughout the body (22) , targeting neurons or cells specifically within the spleen can reduce unintended activation or side effects. There has been a recent surge of research which demonstrates the ability to activate or modulate cells with ultrasound energy (23) (24) (25) (26) (27) (28) There are several studies in rodents showing the ability to modulate the splenic nerve or immune cells within the spleen with ultrasound, to reduce inflammation and cytokine levels (29) (30) (31) (32) (33) . One of the initial reports was in a mouse model of reperfusion injury and kidney inflammation, where ultrasound stimulation of the spleen significantly reduced kidney damage (29, 34) . Recently, our research groups discovered that specific parameters of ultrasound stimulation of the spleen can drive significant anti-inflammatory effects in rodent models of both chronic inflammation (inflammatory arthritis) and acute inflammation (sepsis) (31, 32) , which was shown to be mediated through a similar cholinergic anti-inflammatory pathway accessed through vagus nerve stimulation. Ultrasound is a potentially impactful clinical solution to inflammation as it can be applied non-invasively to the body with a wearable device and with energy parameters already shown to be safe for the human body based on numerous ultrasound imaging applications (35, 36) .

Here, we show the first in-human results of pro-inflammatory cytokine reduction with noninvasive ultrasound stimulation of the spleen. In healthy individuals, a single three-minute administration of splenic ultrasound stimulation significantly inhibits whole blood TNF production upon ex vivo exposure to endotoxin compared to sham controls. In RA patients, we observed that daily splenic ultrasound stimulation results in reduction of blood-borne transcripts encoding for pro-inflammatory markers IL-1β, IL-8, and NFκB, as well as suppresses pathways involved in IL-6 and TNF production. Ultrasound also reduces pathways involved with monocyte migration, contributing to its anti-inflammatory effect. From a safety perspective, circulating immune cell composition does not change with ultrasound treatment, and ultrasound does not inhibit the adaptive immune response in humans. Our additional pre-clinical animal data further demonstrates that in addition to dampening cytokine output and circulating monocyte invasiveness, prophylactic ultrasound activation of the splenic neuroimmune pathway results in enhanced antibody response upon exposure to an inflammatory antigen. Thus, activation of the splenic neuroimmune pathway may provide a low risk therapeutic approach for a broad range of health conditions, due to its pleotropic nature and physiological role in suppressing specific cytokines involved in innate immunity while enhancing the transition to and maintenance of the adaptive immune response (18, 37, 38) . Together, these data suggest great potential for splenic ultrasound as a low-risk clinical therapy for a range of acute or chronic inflammatory diseases without requiring surgery or intake of artificial substances with possible side effects.

Considering that many of the same pro-inflammatory molecules suppressed by ultrasound are implicated as molecular drivers in COVID-19 patients with severe disease, ultrasound also has potential as a non-invasive therapy to combat this pandemic. Drug based suppression of these cytokines for tissue protection comes with the risk of inhibiting viral clearance or increasing susceptibility to bacterial co-infections (39, 40) . Non-invasive ultrasound activation of the splenic neuroimmune pathway may provide an alternative method to combat the cytokine storm without compromising the adaptive immune response in COVID-19 patients, ultimately reducing the high mortality and morbidity rates confronting this worldwide pandemic that currently has limited treatment options.

To investigate how non-invasive ultrasound stimulation of the spleen affects cytokine levels directly in human subjects, healthy participants were recruited into a study designed to investigate effect of different ultrasound parameters and locations of stimulation in the spleen on various immune and metabolic responses during acute stimulation (feasibility study described at clinicaltrials.gov: NCT03548116). Due to the urgency of identifying potential treatment options for COVID-19 patients, data pertaining specifically to ultrasound stimulation of the hilum of the spleen with the most effective intensity parameter in the study (corresponding to one cohort from the study) are presented in this paper.

Participants received pulsed ultrasound stimulation of the spleen for three minutes (2.2 MHz, 1 Hz pulse repetition frequency, 290.4 mW/cm 2 ISPTA) using the GE LOGIQ E9 ultrasound system with the C1-6 probe. An ultrasonographer first identified and targeted the hilum of the spleen, then delivered the pulsed ultrasound stimulus using a modified elastography software setting in the system. Sham stimulation control subjects underwent the same procedure as the stimulated group, including transducer placement and movement of the ultrasound probe to the proper splenic location trajectory, but with no ultrasound energy being transmitted to the spleen from the transducer. Since healthy subjects have low serum TNF levels, we revealed changes in inflammatory status using whole blood challenged with lipopolysaccharide (LPS) ex vivo. This ex vivo cytokine production assay has been previously used to verify splenic neuroimmune activation using implanted vagus nerve stimulators and is a well-established method for non-invasively assessing activation of neuroimmune pathways (18, (41) (42) (43) . A reduction in LPS-induced TNF production in blood sampled from ultrasound stimulated subjects was tested at multiple LPS concentrations (Fig. 1A ) and significant reduction is observed when comparing the ultrasound stimulated group to the non-stimulated controls ( Fig. 1B ; p-value = 0.006 using Wilcoxon unpaired rank-sum test) at the optimal LPS concentration (1 ng/mL; Fig.1A ). These data are consistent with earlier reports using implanted nerve stimulators or pharmaceutical activators of the splenic pathway (18, 42) . They are also consistent with the magnitude of TNF reduction using similar ultrasound stimulation parameters in a previous study activating the neuroimmune pathway in a rodent model of endotoxemia (31) . Further supporting the human results, we also demonstrated that splenic ultrasound stimulation in a rodent model achieves a reduction across a number of proinflammatory cytokines beyond TNF, including IL-6, IFNγ, IL-1β, IL-1α, and IL-12 in splenic lysates (Fig. S1 ), which is consistent with previous reports using implanted vagus nerve stimulators (18, 44) . TNF response after LPS incubation is shown for one ultrasound stimulated subject from samples collected before (0 hours baseline) and after (2 hours post stimulation) ultrasound application. The optimal dose of LPS for measuring the immunomodulatory response to ultrasound was found to be at 1 ng/mL, and this is used for comparing the TNF response between the ultrasound stimulated versus control groups. B) Ultrasound stimulation of the spleen decreases whole-blood LPS-induced TNF release, where blood was obtained from 18 healthy human subjects prior to and then two hours following splenic ultrasound application (stimulated: n=9, control: n=9). The data shown were taken from the 1 ng/ml LPS concentration for each dose response curve for all subjects and samples. p-value was computed using the unpaired Wilcoxon rank-sum test.

Previous animal research demonstrated that ultrasound can significantly reduce inflammation and improve clinical measures in a chronic inflammatory arthritis model (32) . Expanding upon the findings in healthy human subjects in the previous section, we initiated a controlled, randomized clinical trial of splenic ultrasound treatment in RA patients (clinical study described at clinicaltrials.gov: NCT03690466). Participants received ultrasound stimulation to the spleen with the goal of lowering the over-active inflammatory response that occurs during RA flare-ups. Up to 20 RA patients are to be recruited into the study (enrollment is ongoing). Due to the urgency of identifying treatment options for this COVID-19 pandemic, data from single-cell RNA sequencing (scRNA-seq) in peripheral blood mononuclear cells (PBMCs) that has been collected thus far in five ultrasound-treated patients have been analyzed ahead of completion of the study for inclusion into this paper. PBMCs were isolated from whole blood taken from each research participant before ultrasound treatment (day 0, pre-stimulation) and after two weeks of daily 30-minute sessions of ultrasound to the spleen (day 14, post-stimulation). From these 10 samples (subjects = 5, timepoints = 2), a total of 53,343 PBMCs were successfully sequenced from single cells (Table  S1 ). In silico analysis identified 13 cell-types based on gene expression patterns of standardly used marker genes (Fig. S2 ). Participants received stimulation with a 1 MHz transducer (1.2 W/cm 2 ; SoundCare Plus, Roscoe Medical) that was moved continuously across a 5-inch by 5-inch square, centered on the spleen, as identified by an ultrasonographer. Further details on the clinical study design is presented in the Materials and Methods section.

We aimed to identify molecular changes that occur with splenic ultrasound in humans to better understand how this ultrasound treatment can drive an anti-inflammatory response. We observed reduced monocyte expression of key pro-inflammatory cytokine genes encoding for IL-1β and IL-8 from all five subjects post-stimulation compared with pre-stimulation ( Fig. 2A , p-values given for transcripts showing significance of p<0.05). Although this result was determined by analyzing all monocytes, reduction of these two markers appears primarily in CD14+ monocytes as this is the cell type showing predominant expression of those genes (Fig. 2B) . The same analysis in CD8+ T cells show a decrease in transcripts that encode for IFNγ ( Fig. 2A ). The changes in transcript level of these cytokines can only be detected when comparing within the same cell type before and after treatment, within the cell that expresses the cytokine (Fig. S2 ). Consistent with known expression patterns of IL1B and CXCL8 transcripts (encoding for IL-1β and IL-8), we see that these transcripts are only suppressed in the monocytes where they are primarily detected. Likewise, IFNG transcripts (encoding for IFNγ) are detected and found to be reduced in CD8+ T cells (Fig.  S2 ). In our dataset, other cytokines like TNF and IL-6 did not show a significant change in monocytes; however, IL-6 transcripts were detected in only 66 of the 53,343 cells. Therefore, we cannot definitively conclude if there is or is not a change in transcript levels encoding IL-6 with ultrasound treatment.

Since monocytes play a critical role in the innate immune system and the inflammatory response, we further focused our analyses on transcriptional changes that occur in circulating monocytes after ultrasound treatment. Differential expression analysis of all monocytes across the five subjects, pre-and post-ultrasound stimulation, shows 938 differentially expressed genes (p-value<0.05) with 841 of those genes being down-regulated. These results reveal an overall shift in reduction of gene transcripts in monocytes with ultrasound treatment. Additionally, this reduction in transcript levels did not appear as a result of sequencing differences, as we observe no obvious difference in the number of cells sequenced, the number of sequencing reads, or number of average genes detected per cell between monocytes taken from day 0 as compared to day 14 (Table S1 , 'CD14 Mono' and 'CD16 Mono'). Although these gene transcripts are defined as differentially expressed when comparing monocytes from all subjects ( Fig. 2A) , we also observe that for most genes, this down-regulation is consistent across subjects ( Fig. 2C ; columns represent cell-type averages for CD14+ and CD16+ cells for each subject that are similar across columns for many of the genes). showing CD14+ and CD16+ monocytes represented by orange and green dots, respectively. IL-1β and CXCL8 expression on day 0 and day 14 are also mapped on these UMAP diagrams. C) Heatmap displaying the average relative expression (for all cells in specified group) of downregulated transcripts (genes listed on the right, p-value <0.05). Color denotes relative expression for post-ultrasound levels compared to (divided by) pre-ultrasound levels for each subject (represented by columns, n = 5, participant A-E shown left to right) in CD14+ cells or CD16+ cells. Genes encoding for cytokine receptors, components of the JAK/STAT pathway, MAP kinases, and transcription factor (TF) are grouped and labeled on the left of the plot.

These results are quite consistent given the high variability or inhomogeneity in age, sex, physical characteristics, disease properties and past treatments across subjects (participants A-E in Table  S2 correspond to columns 1-5 in Fig. 2C , respectively). Within these down-regulated genes in monocytes, we also observe that many cytokine receptors, such as those for TNF, IL-6, IL-17, IL-13, and IFNγ were reduced with ultrasound treatment (Fig. 2C) . Furthermore, components of signaling pathways downstream of these cytokines are also down regulated, including components of the JAK/STAT pathway, MAP kinases, and pro-inflammatory transcription factor NFκB (Fig.  2C ). Together, these findings demonstrate a consistent and robust reduction in transcripts encoding key pro-inflammatory cytokines and cellular signaling molecules downstream of their receptors after 14 days of daily splenic ultrasound treatment across all five subjects.

Cytokines trigger multiple cellular pathways, which then contribute to systemic inflammation. Our transcriptomic data reveal that many genes known to be regulated by IFNγ and NFκB are subsequently downregulated with ultrasound treatment, further promoting a cascade of suppression across multiple proinflammatory pathways with ultrasound treatment (Fig. 3A) . We sought to determine if the 840 down-regulated genes in monocytes showed statistical enrichment for functional Gene Ontology (GO)terms associated A) Volcano plot showing all detected genes in monocytes, highlighting differentially expressed genes (p-value <0.05) and labeled IFNγ-regulated genes that are down regulated, as well as NFκB-regulated genes that are down regulated as characterized by KEGG pathway gene lists. B) Gene ontology (GO) analysis using Bioconductor package 'topGO', determining functional GO terms significantly enriched within downregulated genes in monocytes. Enriched GO terms associated with cytokine production are shown, where bars represent number of significant genes in a list relative to number of genes that would be expected by chance, and with number of genes contributing to each GO term represented by the shade of blue. pvalues for each term shown is from Fisher exact test. with inflammation. The GO system of classification assigns genes (for differentially expressed gene list) into bins based on their functional characteristics. Gene sets can then be analyzed for GO terms that are statistically over-represented among that set, allowing for a better understanding of which biological processes (e.g., inflammation) may be changed with treatment. Out of the top statistically enriched GO terms for the down-regulated genes, we observe many that contribute to pro-inflammatory cellular processes. Genes suppressed by ultrasound show enrichment of GO terms including "inflammatory response" (p-value = 1.50 Ε-5, Table S3 ), "cytokine-mediated signaling pathway," and "cellular response to cytokine stimulus" (p-values = 9.7 E-5 and 6.7E-6, Fig. 3B ). We also observe enriched GO terms associated with positive regulation of TNF, IL-6, and IL-8 among the down-regulated genes, suggesting that inducing signals for TNF or IL-6 are also suppressed, despite the absences of significant change in TNF or IL-6 transcript levels (Fig.  3B ). These GO term results show that many gene transcripts encoding for cellular inflammatory functions go down after ultrasound treatment.

Within circulating monocytes, we observe ultrasound-treatment associated GO term enrichment of cellular components associated with IL-8 functionality (Fig. 3B) , as well as a robust reduction in IL-8 across subjects (Fig. 4A , columns represent subject cell type averages). IL-8 is a chemokine (chemotactic factor), which stimulates neutrophil movement towards inflammatory sites. Therefore, we further assessed if ultrasound influences other signaling molecules involved with cellular migration (e.g., to sites of inflammation). GO term analysis of down-regulated genes in CD14+ monocytes show enrichment of "positive regulation of cell migration" among the top 15 significantly enriched GO terms. Other GO terms, such as "lamellipodium assembly" (facilitates cell mobility), "ephrin receptor signaling" (promotes cell adhesion), and "actin nucleation" (involved with cell locomotion), all suggest that ultrasound treatment induces down regulation of genes involved with cell migration (Fig. 4B) . Furthermore, genes that more specifically contribute to "monocyte chemotaxis" are down-regulated post-ultrasound stimulation in CD14+ monocytes and many of these genes are consistently reduced across all five subjects (Fig. 4A) , even with quite variable or inhomogeneous patient characteristics (Table S2) .

We sought to determine how ultrasound affects the circulating immune cell repertoire. In comparing the transcriptional profiles (based on principal component analysis and UMAP dimensional reduction) of all circulating peripheral blood mononuclear cells pre-and post-ultrasound stimulation, (Table S4) . C) Percent down regulation range is shown after 14 days of ultrasound stimulation for each gene (represented by dots) in each functional category described previously.

we observe remarkable consistency (Fig. 5A) . Also, the calculated relative percent of each celltype for each subject is displayed, and based on a paired Wilcoxon rank sum test for pre-and poststimulation for each cell type, we observe no significant difference in the relative proportions of percent contribution of any of the 13 cell types (Fig. 5B , statistics shown in Table S4 ). Additionally, when comparing the number of cells sequenced for each cell type per participant there is no significant difference between timepoints (i.e. no technical bias, Table S4 ).

We also determined what degree of suppression occurred after ultrasound stimulation for genes involved in inflammation (genes shown in Fig. 2C, 3A and 4A) . Most genes show a ~5-15% reduction in transcript levels, suggesting a moderate degree of suppression, with IL-8 and genes involved in cell migration showing the highest degree of suppression (>15%, Fig. 5C ). These findings suggest that spleen-targeted ultrasound can potentially provide a way to blunt the elevated release of circulating cytokines and chemokines in inflammatory disorders or viral infections, without disrupting the overall immune cell repertoire.

One concern in treating acute hyperinflammation is that the therapy may suppress the adaptive immune response, and the ability of the immune system to provide pathogen clearance and/or protection from co-infection with a second pathogen. Therefore, we assessed whether non-invasive splenic ultrasound treatment suppresses the adaptive immune response or more specifically antibody production. Our transcriptomic data do not suggest a suppression of the adaptive immune response. In fact, we observe that among genes upregulated in B cells, there is an enrichment for GO terms associated with "B cell activation" (Fig. 6A ). Genes associated with this GO term include transcripts that encode for IgA, IgG, and IgM antibodies. Interestingly, these gene transcripts are upregulated in some of the ultrasound-treated subjects (Fig. 6B) , though with greater variability in results across subjects than was observed for suppression of cytokines and chemokines in monocytes. For example, we observe that for average transcript levels across B cells, transcripts encoding IgG are only upregulated in 2 or 3 subjects (IGHG2 and IGHG4, respectively) out of the 5 subjects. Regardless of the variability across subjects, these data support that ultrasound treatment does not generally inhibit B cell activation and antibody production across patients, and can even potentially enhance antibody production; thus, ultrasound is not expected to compromise the adaptive immune response.

To further investigate the effects of splenic ultrasound stimulation on adaptive immune response and antibody production, as well as the variability we observed across human subjects for transcript levels that encode for IgA, IgG, and IgM antibodies, we performed additional experiments using prophylactic ultrasound stimulation (i.e., ultrasound stimulation prior to antigen exposure) in a rodent model of endotoxemia (31) . We observe that splenic ultrasound stimulation prior to endotoxin exposure (three minutes daily, once a day for 3 days followed by LPS injection on the fourth day) results in a significant increase in IgM and IgG antibody production 24 hours after LPS exposure compared to sham and/or non-LPS controls (p-values listed in Fig. 6C  description) . Furthermore, this enhanced antibody output is dependent on the presence of the toxin/antigen, as there is no observed ultrasound effect on antibody production in the absence of LPS exposure. The increase in antibody transcript levels we observed in some of the human subjects, and not in the others, may be attributed to those participants also having encountered an antigen, such as a cold or other infection that was not identified during the study. This explanation is further supported by a recent report demonstrating that brain-spleen neural pathways are capable of modulating splenic plasma cells differentiation upon presentation of novel T Cell dependent antigens (45) . for naïve animals (no LPS + sham), animals exposed to splenic ultrasound stimulation with no endotoxin (no LPS + ultrasound), animals exposed to the endotoxin but receiving sham stimulation (LPS + sham), and animals receiving both LPS exposure and splenic ultrasound stimulation (LPS + ultrasound). Animals receiving LPS + ultrasound showed significant increases in antibody production when compared to each of the other groups using an unpaired Wilcoxon ranked-sum test for IgM (no LPS + sham, p = 0.00145; no LPS + ultrasound, p = 0.00155; LPS + sham, p =0.01757) and for IgG (no LPS + sham, p =0.02930; no LPS + ultrasound, p = 0.00404; LPS + sham, p =0.04298). No other comparisons using this test showed significance, p < 0.05.

By presenting the first in-human data from two independent studies using different devices and protocols, we have consistently demonstrated that non-invasive ultrasound stimulation of the spleen drives anti-inflammatory effects in the context of both an acute response in healthy subjects and a chronic inflammatory condition. In RA patients after two weeks of daily 30-minute ultrasound treatment to the spleen, there was a reduction in cytokine IL-1β and chemokine IL-8 in circulating monocytes (Fig. 2) . In healthy subjects, we also observed a significant reduction in TNF levels produced by ex vivo LPS-stimulated whole blood, in individuals stimulated with 3 minutes of pulsed ultrasound (Fig. 1B) .

In addition to the reduction of cytokines, we observed a reduction in activation of multiple signaling pathways in circulating monocytes with ultrasound treatment. Cytokines signal by binding to cell surface receptors to set off a cascade of intracellular events. Important cytokines in the context of RA include TNF, IL-6, and IL-1β; their downstream intracellular pathways and necessary signaling molecules include cytokine receptors, components of the JAK/STAT pathway, MAP kinases and NFκB (46, 47) . Many of the transcripts that encode for these critical components are downregulated with ultrasound treatment in RA patients (Fig. 2B) . This finding suggests that in addition to cytokines and their immediate receptors, there is broad suppression of cellular proinflammatory pathways.

Down regulation of these various pro-inflammatory transcripts in RA patients occurs in the context of chronic inflammation. However, these same pathways are also involved in acute inflammatory responses (47) . Genes induced by LPS-stimulated human monocytes include TNF, IL-1, IL-6, and IL-8 (48) . NFκB and associated pathways are also upregulated, and NFκB is activated via phosphorylation during LPS exposure, allowing for a rapid induction of genes so the system can respond in an acute inflammatory setting. These same cellular components are downregulated with ultrasound treatment in our RA patients, suggesting that non-invasive splenic ultrasound could be used clinically to suppress acute inflammation as well.

The discovery that splenic ultrasound can reduce or calm an overactive innate immune response in humans is quite timely. Many of the same key inflammatory cytokines that are reduced with ultrasound stimulation are elevated in COVID-19 patients. Recent studies have revealed that COVID-19 patients consistently produce high levels of IL-1β, IL-8, IL-6, and TNF, which strongly suggests dysregulation in the innate immune response (34) (35) (36) (37) . More specifically, SARS-CoV-2 infected individuals exhibit significantly higher expression levels of IL-1β and IL-6 transcripts in circulating monocytes, as well as IFNγ in CD8+ T cells, shown through PBMC single-cell RNA sequencing of COVID-19 patients (38) . Single cell RNA sequencing has further shown that bronchoalveolar lavage fluid from patients with severe COVID-19 disease have higher levels of IL-8, IL-6 and IL-1β compared to patients with only moderate disease (49) . These molecules are key components of the COVID-19 cytokine storm that function in acute lung injury to recruit neutrophils to alveoli, inducing lung tissue damage (39) . Recently, SARS-CoV-2 infection in children has been associated with a novel multisystem inflammatory disease (MIS-C) resembling toxic shock syndrome or atypical Kawasaki disease (50) . Kawasaki disease also presents with elevated transcripts levels of genes encoding for IL-1, IL-6, IL-8, and IL-17 (51) ; and along with ARDS, is likely to be a condition caused by the COVID-19-induced cytokine storm (52) . Clinical therapies that can suppress these pro-inflammatory cytokines and reduce migration of circulating immune cells to the lungs may be capable of treating patients with moderate to severe cases of COVID-19 to calm the cytokine storm and prevent morbidity and mortality (Fig. 7A) .

Ultrasound has the potential to prevent or improve severe symptoms associated with the cytokine storm in COVID-19 patients. In addition to demonstrating the ability to reduce key proinflammatory cytokines and signaling pathways involved with a hyperactive innate immune response, splenic ultrasound also reduces IL-8 and many components associated with cell migration, specifically monocyte migration (Fig. 4) . These findings are consistent with previous animal studies showing that activation of the cholinergic anti-inflammatory pathway suppresses monocyte migration in mice (46) . Taken together, one proposed mechanism of action for ultrasound treatment is that ultrasound modulates circulating immune cells initially within the spleen via activation of the cholinergic antiinflammatory pathway, which suppresses proinflammatory cytokines and signaling pathways, as well as inhibiting cellular migration, such that there is reduced inflammation at the target site (e.g., at joints in RA or alveoli for COVID-19; Fig.  7A ). Furthermore, data in humans and preclinical animal data show an increase in antibody production with splenic ultrasound.

These results demonstrate an advantageous feature of splenic ultrasound compared to pharmaceutical options for immunosuppression, such as TNF, IL-6, or IL-1 blocking biologics (39, 40) , in that cytokine suppression does not inhibit other functions of the adaptive immune system, such as B cell activity or antibody production. Indeed, splenic ultrasound may enhance the adaptive immune system through the recently discovered brain-spleen neuroimmune pathway that enhances differentiation of antibodyproducing splenic plasma cells (SPPCs) upon antigen activation (45). One limitation of the presented results in the context of COVID-19 or a cytokine storm is that ultrasound stimulation of the spleen may not generate anti-inflammatory effects sufficient to suppress a severe infectious inflammatory response. The anti-inflammatory effects in this paper in response to splenic ultrasound were demonstrated acutely (single 3-minute stimulation) in healthy human subjects or to a moderate extent in RA patients. The question remains whether the antiinflammatory effects observed in healthy subjects or in patients with a chronic inflammation disorder, such as RA, will translate to patients harboring a systemic viral infection, as observed for COVID-19. This question will be answered through a clinical trial that is currently being set up at the University of Minnesota together with GE Research to test this ultrasound approach in COVID-19 patients funded by the Defense Advanced Research Projects Agency (DARPA; Department of Defense).

One key asset of the upcoming COVID-19 study is that GE's cart-based ultrasound system (GE LOGIQ E10) with enhanced targeting capabilities will be used on patients. In the ongoing RA study at the University of Minnesota, a commercially available ultrasound device (SoundCare Plus) was used on patients that did not have targeting capabilities tailored for the spleen. At the onset of that RA study, SoundCare Plus was one of few devices that was already FDA regulated for various stimulation applications; thus, this unit was selected for the RA study. The GE LOGIQ E10 device can be used to better target and stimulate specific locations of the spleen, which the GE team has already demonstrated with strong anti-inflammatory effects in healthy human subjects (Fig. 1B) . Previous animal studies have also demonstrated that stronger anti-inflammatory effects are possible with better focusing of stimulation within the spleen or longer periods on a given target (31, 32) . Furthermore, sufficient activation of the cholinergic anti-inflammatory pathway has been shown to combat acute and deadly cytokine storm conditions in sepsis animal models, and the magnitude of cytokine suppression provided by ultrasound is consistent with the protection provided by implant-based or pharmaceutical methods in these studies (31, 53, 54) .

In parallel with the DARPA-funded study, both GE and a start-up company, SecondWave Systems, are developing wearable or portable ultrasound systems for neuromodulation (Fig. 7B ) that can be positioned over the rib to steer and focus ultrasound energy to different locations of the spleen or other organ targets (Fig. 7C) . Both GE and SecondWave are currently organizing clinical studies at multiples sites worldwide to assess the safety and feasibility of their therapeutic ultrasound devices. Success with these initial clinical studies will propel further development and scale-up efforts for splenic ultrasound technologies to meet the large-scale demand for COVID-19, as well as open up opportunities to treat future viral infections and other inflammatory disorders in combination with or in lieu of biologics.

For the splenic ultrasound study involving healthy human subjects (clinicaltrials.gov: NCT03548116), we provide the data from the two cohorts most relevant to the planned DARPA sponsored COVID study: the sham control cohort (i.e., no ultrasound, with only mechanical pressure over the spleen) and a splenic ultrasound stimulation cohort (i.e., 290.4 mW/cm 2 ISPTA or 1.4 mechanical index (MI) using the shear wave elastography setting on GE LOGIQ E9) that insonified a single site (i.e., the splenic hilum; as identified by a trained ultrasonographer performing the stimulation). The trial was carried out in accordance with International Conference Inclusion and exclusion criteria. To be eligible to participate in the study, an individual must have met the following criteria: aged between 18 and 45 years, be without physical disability or conditions that may make them incapable of undergoing the study procedures or otherwise place them at greater harm, be without significant past medical or surgical histories that would render them at a greater risk of harm, be considered English proficient due to the study requirement to follow verbal commands during the ultrasound session, be considered active as assessed by type of activity (e.g., walking or running) and number of hours a week performing the various activities, be able to attend all study visits at approximately the same time of day, be able to comprehend the study goals and procedures, and able to provide informed consent for participation. See Table S5 for subject characteristics used in the cytokine analysis shown in Fig. 1 .

Study timeline. Subjects underwent a screening visit 1 to 11 days prior to baseline visit to assess eligibility to participate in the study. Eligible individuals that agreed to participate and provided written informed consent then underwent a physical and neurological examination. Women of childbearing potential were asked to provide a urine sample for pregnancy testing, and approximately 21 mL of blood was drawn. On the first protocol visit (day 0), participants again underwent a physical and neurological examination, and medical history review. A baseline blood draw was then collected for cytokine and blood chemistry analysis (~35 mL). The blood draw was performed under sterile conditions using standard venipuncture techniques. Individuals then underwent the ultrasound procedure based on random assignment to one of 7 groups, including the two groups reported herein (i.e., the sham control group, and a stimulated group that received 100% power stimulus applied at a single splenic target location). For ultrasound stimulus delivery, individuals were asked to lie in the right lateral recumbent position with their arms above their heads to expose the splenic region of the abdomen. After ultrasound gel was applied to the region, the ultrasound probe was placed on the participant's abdomen. This procedure was performed for both the sham control group (n=9) and ultrasound stimulated group (n=9). Note that one subject from each group was excluded, in which 10 subjects were initially recruited to the study for each group (see Table S5 ).

Stimulation paradigm. In the ultrasound stimulated cohort, the subject had the dimensions of their spleen assessed (using the B mode imaging setting on the GE LOGIQ E9), and the probe (C1-6) was positioned over the splenic hilum. Stimulation was performed using a modified elastography setting on the LOGIQ E9 operated in two modes: an imaging/targeting mode with short ultrasound pulses (on the order of 1 microsecond) and relatively low acoustic power levels (on the order of 10 mW/cm 2 ), and a stimulation mode (from the clinical shear wave elastography setting) using longer ultrasound pulses (on the order of 1 ms) and higher acoustic power levels (290.4 mW/cm 2 ). Stimulation was performed in twelve 15-second long epochs separated by 15 seconds each, and a total stimulation duration of 3 minutes. The 15 second epochs were performed during breath holds, during which an ultrasonographer was holding the probe in position above the spleen. Before each stimulation period, the ultrasonographer checked the location of the probe using the imaging mode to ensure that all stimulation pulses were on target. The focal landmark used for targeting and stimulation was the hilum of the spleen in the group included in the presented analysis. For the sham control group, the movements and probe contact for the ultrasound procedure were performed by the ultrasonographer, and the total duration of the procedure remained the same as the stimulated group, except that no ultrasound power was applied (i.e., ultrasound output via the probe was turned off). A post-ultrasound blood draw was again performed, and the 2-hour blood draw time-point data is reported herein.

Blood analysis procedures. Prior to the ex vivo cytokine test, the LPS was sonicated for 30 minutes. A 10 mL blood tube (heparin; green cap) was brought into a tissue culture/sterile room and blood was transferred to a 50 mL conical tube. A blinded researcher then diluted the LPS to the test concentrations in 15 pre-labeled tubes containing replicates of the different concentrations of LPS used for blood exposure (dilutions were made to achieve 0, 0.1, 1, and 10 ng/mL of LPS when mixed with blood). Upon mixing the LPS with blood, the final tubes were capped and placed on a rocker within an incubator and the samples were kept at 37 degrees Celsius for 4 hours during incubation. After the incubation, the samples were removed from the incubator, centrifuged at 6000 RPM for 5 minutes, and the plasma supernatant was transferred (via pipette) for storage (frozen prior to analysis). TNF analysis was performed in triplicate from each sample using DuoSet ELISA kit (R&D Systems), as per manufacturer's instructions.

This clinical study is a controlled, randomized, double-blinded trial of short-term (14 days) treatment and is listed at clinicaltrials.gov (NCT03690466) for which a portion of the results are presented in this paper. The study is still ongoing, but the RNAseq data obtained thus far in five ultrasound stimulated patients is presented in this paper for its relevance in revealing the use of splenic ultrasound for potentially treating COVID-19 patients. The protocol, informed consent form, recruitment materials, and all participant materials were submitted and approved by the University of Minnesota's Institutional Review Board (IRB) and monitored by CTSI in accordance with its institutionally approved monitoring plan. The overall objective of the clinical trial is to demonstrate safety and efficacy of spleen ultrasound stimulation in the treatment of RA in 20 participants. For this ongoing study, 17 participants have been recruited, consented, and enrolled with all 17 patients completing the full study. The 17th participant completed the study, however the last 'in-clinic' visit was performed using clinician video conferencing due to the COVID-19 pandemic, while in-person blood sample collection was still able to be performed. Further details for this study can be found on clincialtrials.gov and will be presented in a future publication with complete outcome analyses. The description below only includes relevant methods and RNAseq analyses presented specifically in this paper.

Inclusion and exclusion criteria. To be eligible to participate in the study, an individual must have met the following criteria: 18 years of age or older, carried a diagnosis of seropositive (rheumatoid factor-or cyclic citrullinated peptide antibody-positive) rheumatoid arthritis (RA), and exhibited symptoms or signs of inadequate disease control (either modified HAQ score >0.3 or DAS-28-CRP >3.2). Exclusion criteria included active bacterial or viral infection, pregnancy, malignancy, or inability to provide daily self-care. The patient medical history and characteristics for each of the five subjects presented in this paper is provided in Table S2 .

Study timeline. Participants were assessed during in-clinic visits on days 0 (before treatment began), 3, 7, 10, 14, and 21. Minor flexibility was allowed for scheduling clinic visits outside of holidays for special circumstances. On the first visit, the spleen was imaged by an ultrasonographer and the center of the spleen trajectory was marked on the participant's skin. Participants were instructed on how to self-administer ultrasound treatment to the spleen. The procedure involved sweeping the ultrasound wand continuously across a 5-inch by 5-inch square centered on the spleen mark placed by the ultrasonographer and ensuring full contact was made with the skin during the daily 30 minute stimulation period. Researchers were able to visually confirm that daily treatments were administered through daily online video sessions with the patients. These video sessions took place at about the same time each day for the 14-day treatment period. With each inclinic visit, participants received a joint examination, peripheral joint ultrasound, and blood draw, as well as completing several physical evaluations and questionnaires. Full study details including analysis of all patients and assessments will be presented in a future publication.

Treatment. After an in-clinic training session on Day 0, transcutaneous ultrasound was administered to the spleen for 30 minutes daily for 2 weeks (14 days total) via a portable device (1.2 W/cm 2 ; SoundCare Plus, Roscoe Medical) in the patient's home. The patients were randomized (1:1) to a treatment group or a control (sham) group, in which the latter received the exact same evaluations and stimulation paradigm as the treatment group, except that the device did not deliver any energy from the ultrasound transducer. RA patients and clinical assessors were blinded as to which patients received ultrasound or sham treatment.

RNA-sequencing and statistical analyses. 4mL of blood was collected in a green top LiHep tube from each ultrasound stimulated participant (n=5) on day 0 (before the first splenic ultrasound stimulation was administered) and on day 14 (after the final treatment session). The samples were processed the same day as collection. Peripheral blood mononuclear cells were isolated from whole blood using density gradient centrifugation via SepMate tubes (Stemcell Technologies) and following manufacturer's instructions. PBMCs were then gently frozen and stored at -80 ºC as per 10X Genomics demonstrated protocols GC000039. Following this protocol, once all samples had been collected and frozen, samples were thawed quickly in 20% FBS in PBS, resuspended in 10% FBS, and strained before proceeding to 10X genomics single cell protocol. Samples were sequenced using UMI-based approach from10X Genomics, as previously described (32) and mapped to the human genome.

Transcriptomic analysis was performed using R statistical software, and primary filtering and normalization of each sample was done using Seurat package as previously described (32, 55, 56) . Samples included two timepoints from five subjects and these individual samples were merged for cell type assignment and differential expression analysis. Cell types were assigned using standard marker genes (see Fig. S2 ) as previously described (57) . Differential expression was determined between timepoints for all cells in each cell type using a negative binomial generalized linear model. GO term analysis was performed using the org.Hs.eg.db and topGO packages. GO term enrichment was assessed using the 'elim' algorithm with a Fisher exact test cutoff of 0.01, in which the top 40 significant GO terms in monocytes are shown in Table S3 (58) .

Pre-clinical tests in rodents were performed as previously described (31) . Experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of GE Research. Briefly, a VIVID E9 (GE) ultrasound system was used to perform an ultrasound scan and locate the spleen. A HIFU transducer and system was then positioned on the target area, and a separate ultrasound scan performed using a smaller probe (3S, GE) that was placed within the opening of the HIFU transducer was used to verify alignment of the ultrasound beam with the spleen. Adult Sprague-Dawley rats that were 8-12 weeks old (250-300 g; Charles River Labs) were housed at 25 degrees Celsius on a 12-h light/dark cycle and acclimatized for 1 week, with handling before experiments to minimize the potential confounding measures due to stress response. Water and regular rodent chow were available ad libitum. LPS (0111:B4, Sigma Aldrich) was used to produce a significant state of inflammation in the naïve adult rodents. LPS was administered to animals in the amount of 10 mg/kg, which corresponds to a LD75 dose via intraperitoneal injection.

Treatment. For ultrasound stimulation, animals were anesthetized with 2-4% isoflurane, and the animals were laid on a water circulating warming pad to prevent hyperthermia during the procedures. The region designated for ultrasound stimulation was shaved with a disposable razor and animal clippers prior to stimulation. Ultrasound was applied to the designated area above the spleen using the probe and system described and calibrated from a previous study (31) . An ultrasound stimulus using the previously found optimal stimulation parameters (1.1 MHz, 0.5 ms pulse repetition frequency, 136.36 microsecond pulse length, 568 mW/cm 2 ISPTA) was then applied for 2 minutes, and LPS was immediately injected post-stimulation. Animals were allowed to incubate under anesthesia for one hour, and after incubation the animal was euthanized, and tissue and blood samples were collected. For experiments testing antibody production, an ultrasound stimulus was applied daily (in the morning) for three days prior to LPS injection. On the fourth day, following the three consecutive treatment days, LPS (10mg/kg) was injected intraperitoneally. The animals were then incubated for 60 minutes in accordance with previous studies, and tissue and blood were harvested for analysis of cytokines and antibody production.

Tissue collection. An incision was made starting at the base of the peritoneal cavity extending up and through to the pleural cavity. The spleen was rapidly removed and homogenized in a solution of phosphate-buffered saline, containing phosphatase (0.2-mM phenylmethylsulfonyl fluoride, 5µg/mL aprotinin, 1-mM benzamidine, 1-mM sodium orthovanadate, and 2-µM cantharidin) and protease (1-µL to 20 mg of tissue as per Roche Diagnostics) inhibitors. A targeted final concentration of 0.2-g tissue per mL PBS solution was applied in all samples. After collection of the whole blood, we allowed the blood to clot undisturbed at room temperature for 15-30 minutes. Samples were centrifuged at 1,000-2,000x g for 10 minutes in a refrigerated centrifuge with the resulting supernatant being collected for antibody testing. Samples were then stored at −80 °C until analysis. Splenic tissue lysate was analyzed for cytokine concentrations using ELISA kits for quantifying TNF alpha (Abcam, Ab100785), IFN gamma (Abcam, Ab239425), IL-13 (Abcam, Ab100766), IL-12 (LSBio, LS-F34357), IL-10 (Abcam, Ab100765), IL-2 (Abcam, Ab221834), IL-1 beta (Abcam, Ab100768), IL-1 alpha (Abcam, Ab113350), IL-6 (Abcam, Ab234570) and IL-4 (Abcam, Ab100771) as per manufacturer's instructions for tissue samples. Serum was tested using ELISA kits for quantifying antibody production including IgG (Abcam, Ab189578), and IgM (Abcam, Ab215085) as per manufacturer's instructions. Figure S1 . Modulation of Cytokines with Splenic Ultrasound in Rats Figure S2 . Cellular Transcriptional Signatures . S2C ), and also showing number of reads (counts) and average number of genes for each cell type. We observe that all that all cells have a relatively consistent read depth for the sequencing reaction performed. Also shown are the number of genes out of the human genome that are annotated with that GO term, number of genes from the list of significantly downregulated genes with ultrasound, and the number of genes that would be expected by random chance. Column labeled 'classicFisher' shows p-values from the Fisher's exact test and 'sig_exp' is the number of genes found to be significant in our gene list (decreased with ultrasound in monocytes) compared to the number that might be expected by chance.

C D 3 D C R E M H S P H 1 S E L L G IM A P 5 C A C Y B P G N L Y N K G 7 C C L 5 C D 8 A M S 4 A 1 C D 7 9 A M IR 1 5 5 H G N M E 1 F C G R 3 A V M O 1 C C L 2 S 1 0 0 A 9 H L A − D Q A 1 G P R

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We thank all study participants. We are grateful for the assistance of individuals at the University of Minnesota Genomics Center, especially Jerry Daniel and Emma Stanley. Additionally, we would like to thank Stuart Sealfon and Frederique Ruf-zamojski for their assistance and advice processing PBMC samples for single cell sequencing. This work was supported by the United States Defense Advanced Research Projects Agency (DARPA) Electrical Prescriptions (ElectRx) Program under the guidance of Eric Van Gieson and Gretchen

 Table S6 . Splenic Ultrasound in Healthy Participants: Subject Features. Characteristics and demographic data of 20 enrolled subjects for study of biological effects of ultrasound of the spleen. Ten subjects received full-powered ultrasound treatment at the spleen hilum and another ten subjects were sham controls with the ultrasound device turned off. One of the subjects from the stimulated group was excluded due the selection of the wrong ultrasound imaging protocol by the clinician. One of the subjects from the control group was excluded because their baseline glucose measurement was <50 mg/dL that is considered hypoglycemic.