The Autonomic Nervous System (ANS)-Immune Network in People Living With HIV

The Autonomic Nervous System (ANS)-Immune Network in People Living With HIV | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Autonomic Nervous System (ANS)-Immune Network in People Living With HIV Bridget R Mueller, Mitali Mehta, Maya Campbell, Niyati Neupane, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5504909/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 May, 2025 Read the published version in Clinical Autonomic Research → Version 1 posted 4 You are reading this latest preprint version Abstract Purpose Pre-clinical studies have demonstrated direct influences of the autonomic nervous system (ANS) on the immune system. However, it remains unknown if connections between the peripheral ANS and immune system exist in humans and contribute to the development of chronic inflammatory disease. This study had three aims: 1.) To examine the relationship between IL-6 and the parasympathetic/vagal component of baroreflex sensitivity (BRS-V) in people with HIV; 2.) To determine if the subtype and severity of HIV-autonomic neuropathy (AN) would predict distinct immunotypes; 3.) To compare the burden of non-AIDS-related co-morbidities between immunotypes. Methods 79 adult people with well-controlled HIV underwent a standard battery of autonomic function tests summarized as the Composite Autonomic Severity Score and vagal and adrenergic baroreflex sensitivity (BRS-V and BRS-A). Levels of immune biomarkers were measured in all participants using the Target 96 Inflammation Panel on the Olink proteomics platform and immunotypes were identified using unbiased, non-negative matrix factorization. Mass cytometry (CyTOF) was completed on a subset of participants with and without autonomic neuropathy (N = 10). Results First, we found reduced BRS-V predicted higher levels of IL-6 (p = 0.002). Second, a pro-inflammatory immunotype defined by elevations in type 1 cytokines (IL-6, IL-17) and increased numbers of CD8 + T-cells was associated with autonomic neuropathy characterized by deficits in sympathetic nervous system activity (aOR = 4.7, p = 0.017). This pro-inflammatory immunotype was older with a greater burden of co-morbidities Conclusion Deficits in the parasympathetic/cardiovagal and the sympathetic nervous system are associated with inflammation and disease burden in people living with HIV. Future longitudinal research is needed to examine causality. autonomic nervous system dysautonomia baroreflex neuropathy inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Preclinical studies have demonstrated the importance of the autonomic nervous system (ANS) in regulation of the innate and adaptive immune system. However, our knowledge of the ANS-immune network in humans is limited. Decades of research have established that autonomic neuropathy (AN) is prevalent among people with HIV 1 and HIV-AN is associated with significant morbidity and mortality. 2 More recently, AN has been found in patients with chronic inflammatory disorders including Long Covid (LC) syndrome 3 , 4 , myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) 5,6 , and autoimmune disorders including multiple sclerosis. 7 – 9 Despite the significant prevalence of AN in chronic inflammatory disorders, the etiology of the comorbidity is unknown, and a greater understanding of the ANS-immune network is needed. In vitro and studies in animals have demonstrated that the extensive peripheral ANS, comprised of sympathetic efferents, parasympathetic (i.e. vagal) efferents, and sensory afferents has the ability to exert targeted, integrated, and rapid modulation of immune signaling through inflammatory reflexes. 10 Lymphoid organs are highly innervated by sympathetic fibers and the sympathetic neurotransmitter norepinephrine (NE) regulates immune cell production and release of cytokines via α- and β-adrenergic receptors on located on immune cells. At high concentrations, NE released by SNS efferents has an inhibitory effect on immune cells through β-adrenergic receptors, 11 while NE released by the sympathetic adrenal medullary (SAM) axis circulates at lower concentrations, preferentially activating pro-inflammatory, high-affinity α-receptors. 12 , 13 The influence of parasympathetic/vagal activity is primarily anti-inflammatory and often referred to as the cholinergic anti-inflammatory pathway (CAP). 14 Pre-clinical studies have established that activation of the CAP results in a reduction of inflammatory cytokine release, including interleukin-6 (IL-6), from splenic macrophages.. 15 , 16 In vitro studies have demonstrated that the principal neurotransmitter of the parasympathetic nervous system, acetylcholine, released by vagal efferents, has anti-inflammatory effects on immune signaling. In a lipopolysaccharide-stimulated (LPS) human macrophage culture, the application of acetylcholine attenuated the release of pro-inflammatory TNFα, IL-6, and IL-18, but did not influence levels of the anti-inflammatory cytokine IL-10. 23 Our knowledge of ANS regulation of the immune system in humans stems mainly from observational studies focused on parasympathetic/vagal function. Cardiac measurements indicative of lower resting vagal activity are present in patients with inflammatory bowel disease (IBD) as well as rheumatoid arthritis (RA) and neuropsychiatric conditions. 17 , 18 In addition, vagal nerve stimulation has been associated with reduced levels of pro-inflammatory cytokines in patients with Sjogren’s syndrome and Crohn’s disease. 19 , 20 Due to the complexity of the sympathetic nervous system and the methodological challenges associated with its measurement, the influence of sympathetic activity on immune signaling pathways in humans remain largely unexplored. This leaves a significant gap in our understanding of the ANS-immune network, as branches of the ANS do not act independently, but modulate immune signaling through an interconnected network. 10 , 15 Our study had three goals. As pre-clinical studies have demonstrated the importance of IL-6 in the CAP 10 and IL-6’s association with increased morbidity and mortality in people with HIV 21 , 22 , we first investigated the relationship between IL-6 and the parasympathetic/vagal component of baroreflex sensitivity (BRS-V) in people with HIV. We chose to measure BRS-V because this reflex relies on caudal vagal circuitry common to the CAP. 23 Our second goal was to holistically examine the ANS-immune network and determine if the type (i.e. parasympathetic/vagal, cardiovascular sympathetic, and non-cardiovascular sympathetic) and severity of autonomic dysfunction was associated with distinct immune phenotypes identified by unsupervised non-negative matrix factorization (NMF) clustering analyses performed on a panel of 96 biomarkers of inflammation. Finally, as AN is associated with medical morbidity in people with HIV, 2 we sought to compare the burden of non-AIDS-related co-morbidities between immunotypes. Given the availability of medications and devices that modulate both the ANS and the immune system, we hope a greater understanding of the ANS-immune network will rapidly translate into therapeutic studies. Materials and Methods 2.1 Study Design and Patient Population. This is a cross-sectional observational study. Participants were recruited from a primary care clinic network within the Mount Sinai Health System in New York City which provides primary care to more than 10,000 people living with HIV. Eligible participants were identified by pre-screening clinic providers’ schedules and requesting approval from providers prior to contacting potential participants. Included participants were at least 18 years of age with a history of well-controlled HIV infection (plasma RNA load of ≤ 100 copies/ml for 3 months prior to enrollment) who were on a stable combination of antiretroviral treatment (CART) for at least 3 months. Patients with another diagnosis known to cause autonomic dysfunction (e.g., diabetes) not able to complete required autonomic testing (e.g., participant must be able to stand) or taking substances that impact the autonomic nervous system (e.g. urine drug testing positive for stimulants) were excluded. All procedures were performed in accordance with a protocol approved by the Institutional Review Board of the Icahn School of Medicine at Mount Sinai (ISMMS) and all participants provided written informed consent. 2.2 Autonomic testing procedures : Autonomic function tests (AFTs) are a standard battery of non-invasive tests (WRMed) which include sudomotor testing (QSWEAT), heart rate response to deep breathing, Valsalva maneuver (VM), and tilt table testing. The QSWEAT is performed by placing a capsule containing acetyl choline (ACh) on the skin in four standardized locations (forearm, proximal leg, distal leg, and foot). The capsule is attached to an automated system which delivers a small continuous electrical stimulus to the capsule causing iontophoresis of ACh into the skin, which triggers a reflexive sweat response collected by the capsule. The evoked sweat volume is measured and compared to standardized values. A non-invasive continuous beat-to-beat blood pressure (BP) monitoring device is attached to the participant’s finger and a 3-lead surface electrocardiogram and respiratory monitor are attached to the chest. BP, heart rate (HR), and respirations are recorded during the VM (forced exhalation to a pressure of 40 mmHg for 15 seconds), standardized paced deep breathing (HRDB), and a 10-minute head-up tilt test. 2.3 Calculation of autonomic indices : The above-described procedures are used to calculate the Composite Autonomic Severity Score (CASS), which is an age- and sex-adjusted summary score reflecting overall autonomic function and is the sum of three sub-scores. The sudomotor (i.e. peripheral, non-cardiovascular sympathetic) sub-score uses data from the QSWEAT, the parasympathetic/vagal sub-score is based on changes in HR during deep breathing and VM, and the adrenergic (i.e., cardiovascular sympathetic) sub-score is based on BP changes during VM and tilt table testing. Given the medical complexity of our patient population, our lab uses the stringent threshold of a total CASS ≥ 3 to define AN; a sub-score of 1 defines mild dysfunction and a sub-score ≥ 2 identifies moderate to severe dysfunction. 24 Baroreflex sensitivity (BRS) was calculated as previously described. 25 Briefly, VM data is visually inspected by a trained, blinded technician. BRS-V is a measure of the compensatory cardiac response to a decrease in BP evoked during the forced expiration against a closed glottis and is calculated by dividing the change in RR interval during phase 2E of the VM by the change in systolic blood pressure. It is a continuous measurement expressed as milliseconds/mmHg. BRS-A is expressed in mmHg/second, and it is calculated by dividing the change in systolic blood pressure during phase 3 by the time required for SBP to recover following the release of VM. 2.4 Medical history and patient-reported outcome measures : Medical history and concomitant medications were obtained through participant interview and review of the electronic health record (EHR). To control potentially confounding influences of medications and comorbidities, several indices were included in multivariable analyses. First, as acetylcholine is a main neurotransmitter of the ANS, an anticholinergic burden (ACB) medication score was determined for all participants. Second, a Charlson Comorbidity Index was calculated (modified to exclude HIV/AIDS). 26–28 Finally, as there is significant overlap between the limbic neural circuitry that underlies emotion regulation and rostral pathways of the central autonomic network (CAN), participants completed the Hospital Anxiety and Depression Scale (HADS) 29 and the Perceived Stress Scale-14 (PSS-14). 30 2.5 Biospecimen Collection and Proteomics Analysis : Whole blood was collected in EDTA tubes followed by isolation of peripheral blood mononuclear cell (PBMC) and plasma, as described previously. 31 Serum was obtained concomitantly from serum-separator tubes. At our institution’s Human Immune Monitoring Center (HIMC), a shared core research facility, plasma IL-6 was measured as part of the Target 96 Inflammation Panel on the Olink proteomics platform (Uppsala, Sweden) as previously published. 31 2.6 Cytometry by time of flight (CyTOF) : CyTOF was performed on PBMC samples from participants with AN (CASS ≥ 3) and without (CASS ≤ 1) selected purposively to maximize the difference in CASS between the groups while balancing sex and age (N = 5 per group) and without knowledge of cytokine profiles. Briefly, cell counts were performed on the Nexcelom Cellaca Automated Cell Counter (Nexcelom Biosciences) and cell viability was measured using Acridine Orange/Propidium Iodide viability staining reagent (Nexcelom). After washing cells in Cell Staining Buffer (CSB) (Fluidigm) Fc receptor blocking (Biolegend) and Rhodium-103 viability staining (Fluidigm) were performed simultaneously with surface markers for 30 minutes at room temperature. Cells were subsequently washed twice in CSB and then palladium barcoding was performed on each sample using the Cell-ID 20-Plex Pd Barcoding Kit (Fluidigm) following manufacturer’s instructions and pooled together. The pooled sample was fixed with 2.4% PFA (Electron Microscopy Sciences) followed by labeling with 125nM Iridium-193 (Fluidigm) and 2nM Osmium tetroxide (EMS) for 30 minutes at room temperature. Immediately prior to data acquisition, samples were resuspended at a concentration of 1 million cells per ml in Cell Acquisition Solution containing a 1:20 dilution of EQ Normalization beads (Fluidigm). The samples were acquired on a Helios Mass Cytometer (Fluidigm) equipped with a wide-bore sample injector at an event rate of < 400 events per second. Prior to analysis, routine data normalization (Fluidigm software) and sample demultiplexing were undertaken. 2.7 Statistical analysis : All data were stored in REDCap. Descriptive statistics including frequencies, percents and medians with interquartile range were calculated as appropriate. Spearman rank correlation was performed to investigate the relationship between IL-6 and BRS-V. While CASS normative scoring is adjusted for age and sex, BRS is not. Therefore, multivariate logistic regression adjusting for age and sex was performed for analyses involving BRS. Kruskal-Wallis or Chi-square compared continuous and categorical variables between the four immunotypes, respectively. Bonferroni correction was applied to analyses involving multiple comparisons. Immunotypes were defined using the R package for nonnegative matrix factorization (NMF) as previously described. 32 Normalized protein expression of Olink inflammatory markers were compared between immunotypes. STRINGdb R package v2.16.4 was used for gene ontology (GO) enrichment analysis to elucidate key biological processes associated with immunotypes. The significance of GO terms was determined based on P-value (< 0.001) and FDR (< 0.05). To account for multiple testing and reduce the likelihood of false positives, we applied the Benjamini-Hochberg test for corrections. All analyses were conducted using SPSS version 28 and R 4.3.0. Results 3.1 Study population demographics, clinical characteristics, and autonomic function Participant demographics and medical characteristics are summarized in Table 1. Participants (N = 79) had an average age of 51.6 years (range: 25–73 years of age) and approximately three quarters were male. The majority had long-standing HIV with a self-reported mean of 22 years. African American was the most common race/ethnicity (48%), while 17% of participants were Hispanic/LatinX, 17% were non-Hispanic/LatinX white and 17% identified as other. With regard to medication, the mean ACB score for participants was 0.3 (SD = 0.8) with a range of 0 to 4. Regarding medical comorbidities, the median Charlson was 1.0 (IQR 0–1), which aligns with previously reported values in people living with HIV. 33 Hypertension and hyperlipidemia were the most common comorbidities in our population (38.0% and 31.7% respectively; Table 1). 76/79 participants completed autonomic nervous system testing. The total CASS ranged from 0–6 with a median score of 2.0. AN was common (44.7%) in our study population. Examination of CASS sub-scores showed that 42% of all participants had adrenergic (i.e., cardiovascular sympathetic) sub-score abnormalities, 57% had sudomotor (i.e. non-cardiovascular sympathetic) abnormalities, and 49% had cardiovagal (i.e., vagal/parasympathetic) dysfunction. In patients with AN, dysfunction across multiple domains of ANS function was common, with dysfunction in two domains present in 58.9% of participants and 29.4% demonstrating dysfunction in all three domains. 3.2 Increased IL-6 In Patients with Parasympathetic/Vagal Dysfunction We first explored the relationship between IL-6 and parasympathetic/vagal function. Spearman rank correlation revealed a significant univariate correlation between IL-6 and BRS-V in the expected direction with reduced vagal function correlating to higher IL-6 (Spearman’s rho = -0.352, p = 0.002; Fig. 1 ). This was confirmed with multivariate, stepwise, linear regression with IL-6 as the outcome variable and BRS-V, age, ACB score, CD4+, sex, Charlson score, and diagnoses of anxiety and depression as the predictors; the final model retained BRS-V (p = 0.012) and age (p = 0.028). 3.3 Unsupervised Clustering Identified a Pro-inflammatory Immunotype with Older Participants, and a High Prevalence of AN and Burden of Co-morbid Illness Next, we assessed the profile of inflammatory proteins using unbiased, non-negative matrix factorization (NMF) and found four clusters or immunotypes, with distinct differentially expressed protein expression profiles, as demonstrated in the resulting heatmap (Fig. 2 A). These immunotypes differed with respect to prevalence and severity of AN, age, as well as burden of co-morbid illnesses (Tables 1 and 2). Of note, sex, CD4 + count, HADS scores, ACB score, and PSS-14 scores did not differ between the four immunotypes (Table 1). Immunotype 1 was defined by an upregulation of pro-inflammatory biomarkers including interleukins (IL-6, IL-17), and members of the TNF families (IL-18, TNF-β, TNFRSF9, OPG) which are regulated by the CAP. Immunotype 1 also had increased levels of interleukins involved in the expansion and activation of CD8 + T- cells, including IL-12B, IL-17c, and IL-10RB, and decreased levels of proteins that act to inhibit T-cell expansion including CASP-8. 34 (See Supplemental Table 1). GO analyses demonstrated an enrichment in proteins involved in T-helper 1 cell cytokine production, as well as the migration and chemotaxis of several immune cell subtypes (Fig. 3 A). Immunotype 1 was six years older than the study population overall (60.5 years versus 53.5 years) and had a higher median Charlson Comorbidity Index compared to other immunotypes. The most prevalent medical comorbidities were hypertension (47.6%) and hyperlipidemia (42.9%). Immunotype 1 also had the highest prevalence of AN (70%). In multivariate logistic regression participants with AN were almost five times more likely to be immunotype 1 than immunotypes 2–4 (aOR = 4.7, p = 0.017) after adjusting for age, sex, ABC score, Charlson Index, and CD4 + count. All participants with moderate to severe AN (CASS > 4) were in immunotype 1 and no participants with a CASS of zero were in immunotype 1. Cardiovascular sympathetic nervous system deficits defined this immunotype as they were present in three-quarters of participants in immunotype 1, compared to just 20–30% in other immunotypes. (Table 2, Fig. 4 ; p = 0.001) Immunotype 2 also demonstrated a pro-inflammatory profile. Examination of normalized protein expressed showed that many of the immunotype 1 defining proteins, including IL-18 and TNF-β, were upregulated in immunotype 2, though to a lesser extent (Supplemental Table 1, Fig. 2 ). Similarly, while NMF analyses identified the pro-inflammatory vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), IL-12, and Fms-related tyrosine kinase 3 ligand (FLT3LG) as immunotype 2 defining proteins, these proteins were also upregulated in immunotype 1, though to a lesser extent. Immunotype 2 also demonstrated an increased level of anti- inflammatory cytokines and proteins indicative of cell apoptosis and the negative regulation of cytokine production (e.g. IL-10, TNFSF10) which were not upregulated in immunotype 1 (Fig. 2 ). These results aligned with pathways identified in GO enrichment analyses (Fig. 3 B). Immunotype 2 was significantly younger than immunotype 1 (47.0 versus 60.5 years) and had a significantly lower Charlson Comorbidity Index than immunotype 1 (0.5 versus 2.0). Immunotype 2 also had the shortest HIV disease duration, though this was not a statistically different result. Immunotype 2 had a lower prevalence of AN (44.8%) than immunotype 1, which was due to a reduced prevalence of adrenergic deficits (20% versus 75%). Immunotype 3 had a different immune signaling profile compared to immunotypes 1 and 2. The pro-inflammatory proteins elevated in immunotypes 1 and 2 were not elevated in immunotype 3 (Supplemental Table 1) and similar to immunotype 2, GO enrichment analysis demonstrated upregulation of proteins involved in the negative regulation of cytokine production and differentiation of immune cells (Fig. 3 C). Defining proteins identified by NMF included the glial derived protein family (GDNF and NTN), neurotrophin 3 (NTF3) and IL-4, a cytokine involved in the differentiation of TH cells into TH2 cells. 35 Demographically, the age and comorbidity burden of immunotype 3 did not differ from the study population average. However, immunotype 3 had significantly more African Americans than other immunotypes (87.5%). Immunotype 4 also had a lower inflammatory profile compared to immunotypes 1 and 2, though there was significant upregulation of several pro-inflammatory proteins related to immune cell chemotaxis including CXCL11 and monocyte chemoattractant protein-1 (MCP-1), which distinguished it from immunotype 3. (Supplemental table 1). GO analysis identified enriched expression of pathways involved in intracellular signaling and positive regulation of immunoglobulin secretion (Fig. 3 D). Immunotype 4 had the highest prevalence of solid tumors, HSV, and asthma compared to other immunotypes, although this was not statistically significant. Approximately one-third of participants in immunotype 4 had AN (36.4%) but, like immunotype 3, the majority of autonomic dysfunction was mild (Fig. 4 ). 3.4 CyTOF: To gain a greater understanding of how AN alters the immune profile, we performed a cellular profile analysis using high-parameter CyTOF, comparing the cellular composition between patients diagnosed with and without AN. As described in the methods, participants were selected purposefully to maximize the difference in CASS, and without knowledge of their cytokine profiles. Interestingly, we found that all patients in the AN group belonged to immunotype 1. CyTOF results showed an increased abundance of CD8 + T-cells in patients with AN compared to those without AN, which was particularly pronounced for the terminally differentiated effector memory subtype of the CD8 + T cells (Fig. 5 ). Discussion We utilized comprehensive ANS testing and a broad-scale proteomic approach to assess the ANS-immune network in 79 people living with HIV. We found autonomic neuropathy (AN) was prevalent (44.7%) and predicted a pro-inflammatory phenotype characterized by elevations in type-1 cytokines, a predominance of CD8 + T-cells, and a higher burden of co-morbid illness in people living with HIV. These findings provide novel evidence for the clinical significance of parasympathetic/vagal and sympathetic pathways in the regulation of immune signaling. This is the first study to demonstrate that diminished activity of caudal parasympathetic/vagal circuitry, measured by vagal baroreflex sensitivity, predicted elevated IL-6 in people with HIV. IL-6 plays a pivotal role in vascular inflammation, endothelial dysfunction, and adverse cardiovascular events. 36 , 37 Yet, despite pre-clinical studies demonstrating IL-6’s regulation by the CAP, our understanding of this pathway in humans is limited. The majority of studies examining the relationship between parasympathetic/vagal activity and systemic inflammation use resting heart rate variability (HRV) measurements. 17 , 18 , 37 , 38 However, resting HRV is associated with the activity of prefrontal-amygdala pathways 39 and is decreased in stress-related mood disorders. 40 Our own lab found reduced resting HRV in patients with normal peripheral vagal pathways. 41 We found only one study that used a reflexive measure of vagal activity to assess the relationship between plasma IL-6 and caudal parasympathetic/vagal circuitry. In young patients with type I diabetes without AN, lower HRDB correlated to higher levels of plasma IL-6. 42 Thus, our results provide novel support for the relevance of the CAP in patients with chronic inflammatory AN. Unsupervised NMF clustering analysis revealed distinct immunotypes in people with HIV, supporting previous work that has demonstrated low and high inflammatory subgroups in people with HIV. 43 Immunotype 1 was significantly older and had a higher burden of comorbid illness compared to immunotype 2. While both immunotypes 1 and 2 demonstrated upregulation of pro-inflammatory proteins associated with Type 1 T-cell expansion, immunotype 2 also had an enrichment of proteins involved in the negative regulation of inflammation and apoptosis, which was supported by GO analysis. The upregulation of these inhibitory proteins distinguished immunotype 2 from immunotype 1. The enrichment of CD8 + T-cells in patients with AN supports the proteomics analyses showing an upregulation of plasma immune biomarkers associated with proliferation of Type 1 T-cells in immunotype 1. The association of HIV-AN with a greater abundance of CD8 + T-cells is particularly relevant given that low CD4/CD8 ratio has been implicated in multiple poor outcomes in people with HIV including neurocognitive disorders, malignancy, and cardiovascular disease. 44 Surprisingly, the pro-inflammatory immunotype 1 did not display a greater prevalence or severity of parasympathetic/vagal dysfunction. Instead, pathology in the sympathetic nervous system distinguished immunotype 1 from the younger and healthier immunotype 2. The increased prevalence of cardiovascular sympathetic deficits was particularly striking (75% in immunotype 1 versus 20% in immunotype 2). Non-cardiovascular sympathetic deficits, as measured by the sudomotor sub-score, were also more common in immunotype 1, indicating widespread sympathetic dysfunction. Pre-clinical studies provide an anatomic and physiologic basis of the anti-inflammatory impact of the sympathetic nervous system. 11 In vitro studies have demonstrated that local release of NE from sympathetic efferents inhibits the production and release of pro-inflammatory cytokines through actions at β-adrenergic receptors located on the surface of immune cells. 11 While we could not directly assess the sympathetic efferents innervating organs of the immune system, in more advanced HIV-AN with evidence of both cardiovascular and non-cardiovascular sympathetic impairment (as immunotype 1 displayed), it would be unlikely that they would be selectively spared. 45 Moreover, loss of sympathetic innervation of lymph nodes has been demonstrated in Simian Immunodeficiency Virus (SIV) SIV models. 46 Future studies should examine if organ-specific deficits in post-ganglionic sympathetic efferent activity are present in people living with HIV. The significant age difference between immunotype 1 (median 60.5 years) and 2 (median 47.5 years) warrants discussion. Determining the influence of aging on HIV-AN progression may be confounded by the common correlation between age and duration of HIV disease and the fact that younger people with HIV did not experience the early HIV epidemic (i.e., lack of treatment followed by neurotoxic treatments). Interestingly, disease duration did not significantly differ between immunotypes 1 and 2 and suggests that aging may contribute to deficits in cardiovascular sympathetic activity that characterize immunotype 1. We hypothesize that with time, the autonomic, immune and clinical characteristics of immunotype 2 may evolve to resemble those of immunotype 1. Clinical and pre-clinical studies indicate aging comprises peripheral nerve structure and function through mechanisms that involve inflammation, and thus may be accelerated in the context of the pathologic inflammation of chronic HIV. 47 , 48 Unmyelinated, type C fibers of the sympathetic nervous system may be more vulnerable to aging than type A and B vagal efferent projections. 49 Longitudinal research examining the progression of HIV-AN and its influence on the immune network is needed to explore this hypothesis. The diminished prevalence of AN in immunotype 3 may have contributed to its lower inflammatory profile. However, it is important to note that a significantly greater proportion of immunotype 3 identified as African American (87.5%) and while isolating the biological influence of race on the immune system is complicated by cultural and psychosocial influences, there is evidence for race-based differences in immune signaling which may confound conclusions regarding the ANS’s influence on immune signaling. 50 Similarly, immunotype 4 had a higher prevalence of solid tumors, HSV, and asthma, and thus, AN is unlikely to be the primary influence on immune signaling. There are certain limitations to our study. First, the cross-sectional design and a small sample size prohibit establishing mechanistic causation and larger, longitudinal studies are needed. However, this work does provide important support for a pre-clinical literature that demonstrates direct modulation of the immune system by the ANS. Second, while female representation in our study aligns with HIV disease prevalence, the lower enrollment makes it difficult to examine the likely influence of sex differences on ANS-immune function. Finally, additional tests of autonomic dysfunction such as catecholamine plasma levels in response to standing or intraepidermal nerve fiber density on skin biopsy were not obtained. Therefore, to acknowledge this limitation, we chose a higher CASS threshold to identify autonomic neuropathy. In conclusion, our results provide important evidence that the cholinergic anti-inflammatory pathway (CAP), established by pre-clinical studies, may be relevant to the pathology of chronic inflammatory disorders. In addition, these data demonstrate the importance of the sympathetic nervous system in the development of a pro-inflammatory immune signature associated with a higher burden of co-morbid disease and suggest that longitudinal examination of the time course of HIV-AN progression may provide important insight regarding the influence of aging on this process. Finally, our findings illustrate that a comprehensive evaluation of the ANS-immune network provides a greater understanding of core mechanisms that lead to immune system dysregulation associated with increased morbidity and mortality in people with HIV and provide a rationale for future research to develop therapeutic strategies focused on modulation of the sympathetic and parasympathetic/vagal nervous system. Declarations Acknowledgments: This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; R01DK122853) and the National Institute of Health (NIH) Helping to End Addition Long Term (HEAL: K12NS130673). This work was also supported in part through the computational and data resources and staff expertise provided by Scientific Computing and Data at the Icahn School of Medicine at Mount Sinai and supported by the Clinical and Translational Science Awards (CTSA) grant UL1TR004419 from the National Center for Advancing Translational Sciences. The authors have no conflicts of interest relevant to this work. References Robinson-Papp J, Sharma S, Simpson DM, Morgello S. Autonomic dysfunction is common in HIV and associated with distal symmetric polyneuropathy. J Neurovirol . Apr 2013;19(2):172-80. doi:10.1007/s13365-013-0160-3 Kwon PM, Lawrence S, Figueroa A, Robinson-Papp J. 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Apr 2021;7(2):e571. doi:10.1212/NXG.0000000000000571 Tables Table 1: Participant Demographics Overall N = 79 Immunotype 1 N = 21 Immunotype 2 N = 29 Immunotype 3 N = 16 Immunotype 4 N = 11 p-value Age, years* 53.5 (42.0, 61.5) 60.5 (52.8, 63.3) 47.0 (18.5) 52.5 (21.25) 54.0 (27.0) 0.043 Sex, male 58 (73.4) 14 (66.7) 22 (75.9) 11 (68.8) 8 (72.3) 0.778 Race ethnicity 0.017 African American 38(48.1) 6 (29.0) 15 (51.7) 14 (87.5) 2 (18.2) Hispanic/LatinX 14(17.7) 7 (33.3) 5 (17.2) 0 (0) 1 (9.1) White 13(16.5) 4 (19.0) 4 (13.8) 1 (6.3) 4 (36.4) Other 14(17.7) 3 (14.3) 5 (17.2) 1 (6.3) 3 (27.3) Latest CD4+ count (cells/mm 3 ) 578.0 (463.5,815.3) 584.5 (244.0,837.5) 640.5 (496.3, 851.3) 547.0 (437.3, 855.3) 521.0 (350.0, 758.0) 0.496 Time since HIV diagnosis, years 23.0 (15.0, 32.0) 24.8 (19.4, 30.1) 21.1 (17.3, 25.0) 24.3 (19.2, 29.4) 25.0 (19.4, 30.5) 0.521 HADS Anxiety 7.1 (2.6, 11.6) 7.6 (5.1,10.0) 6.6 (5.1, 8.0) 7.8 (5.2,10.5) 7.0 (3.7, 10.3) 0.818 HADS Depression 4.6 (0.81, 8.39) 5.4 (3.5,7.2) 4.1 (2.8,5.5) 5.2 (3.2,7.1) 3.8 (0.9, 6.5) 0.541 PSS-14 Score 16.5 (9.0, 24.0) 16.3 (12.5, 20.0) 14.9 (12.4, 17.4) 16.9 (12.3, 21.6) 15.8 (9.4, 22.3) 0.391 CCI Score 1.0 (0.0, 2.0) 2.0 (1.0, 5.0) 0.5 (0.0, 2.0) 1.0 (0.0, 2.0) 1.0 (0.0, 6.0) 0.013 ACB Score 0.3 (-0.5,1.1) 0.7 (0.1 1.3) 0.2 (0.0, 0.4) 0.2 (0.0, 0.4) 0.2 (0.0, 0.4) 0.798 Comorbidities Hypertension Hyperlipidemia CAD Syphilis history HSV Asthma Obesity Osteoarthritis 30 (38.0) 10 (47.6) 13 (44.8) 5 (31.3) 2 (18.2) 0.329 25 (31.65) 9 (42.9) 8 (27.6) 5 (31.3) 3 (27.3) 0.570 3 (3.8) 2 (9.5) 1 (3.4) 1 (6.3) 0 (0) 0.387 19 (24.1) 3 (14.3) 11 (37.9) 2 (12.5) 3 (27.3) 0.225 9 (11.4) 4 (19.0) 1 (3.4) 1 (6.3) 3 (27.3) 0.086 14 (17.7) 4 (19.0) 5 (17.3) 2 (12.5) 3 (27.3) 0.819 5 (6.3) 3 (14.3) 1 (3.4) 1 (6.3) 0 (0) 0.308 7 (8.9) 4 (19.0) 2 (6.8) 0 (0.0) 1 (9.1) 0.209 Solid tumor 9 (11.4) 3 (14.3) 2 (6.8) 1 (6.3) 3 (27.3) 0.262 Unless otherwise stated, data are summarized as mean (95% confidence interval) or N (% of column). *, median (interquartile range). Each subscript letter denotes a subset of immunotype categories whose column proportions do not significantly differ from each other at the 0.05 level. Abbreviations: CD4: Cluster of Differentiation 4; HADS: Hospital Anxiety and Depression Scale; PSS-14: Perceived Stress Scale; ACB: Anticholinergic Burden; CAD: Coronary Artery Disease; HSV: Herpes Simplex Virus Table 2: ANS activity of immunotypes Overall N = 76 Immunotype 1 N = 20 Immunotype 2 N = 29 Immunotype 3 N = 16 Immunotype 4 N = 11 p-value Total CASS 2.0 [1.0,3.0] 3.5 [2.0, 4.0] 2.0 [0.0, 3.0] 1.0 [1.0, 2.0] 2.0 [0.0, 3.0] 0.005 a < 0.001 b Autonomic neuropathy (CASS ≥ 3) 34 (44.7) 14 (70.0) 13 (44.8) 3 (18.8) 4 (36.4) 0.020 a < 0.001 b Sudomotor CASS 1.0 [0.0,2.0] 2.0 [0.0, 4.5] 0.0 [0.0,2.0] 1.0 [0.0, 2.0] 0.0 [0.0, 2.0] 0.265 a 0.048 b Adrenergic CASS 0.0 [0.0,1.0] 1.0 [0.3,1.8] 0.0 [ 0.0, 0.0] 0.0 [0.0, 1.0] 0.0 [0.0, 1.0] < 0.001 a < 0.001 b Cardiovagal CASS 0.0 [0.0,1.0] 1.0 [0.0, 2.0] 1.0 [0.0, 1.0] 0.0 [0.0, 0.75] 0.0 [0.0, 1.0] 0.161 a 0.198 b BRS-V, ms/mmHg 8.0 [0.0,17.7] 3.9 [1.4, 17.5] 8.4 [3.5, 13.3] 8.1 [6.0, 14.3] 8.0 [4.1, 13.8] 0.504 a 0.190 b BRS-A, ms/mmHg 13.1 [3.1,23.1] 10.1 [0.0, 21.5] 13.3 [8.0, 19.7] 13.6 [8.7, 18.2] 14.9 [11.3, 18.0] 0.630 a 0.248 b Data presented is median [1q, 3q] or N (% of column) Abbreviations: BRS-A: adrenergic baroreflex sensitivity; BRS-V: vagal baroreflex sensitivity a, comparison across four immunotypes b, binary comparison between immunotype 1 and immunotypes 2-4 Supplementary Files supplementaltables.docx Cite Share Download PDF Status: Published Journal Publication published 10 May, 2025 Read the published version in Clinical Autonomic Research → Version 1 posted Reviewers agreed at journal 25 Nov, 2024 Reviewers invited by journal 25 Nov, 2024 Editor assigned by journal 23 Nov, 2024 First submitted to journal 22 Nov, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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interleukin-(IL-6).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5504909/v1/cb54b3c920a1dd8b6af5c52e.png"},{"id":72300254,"identity":"3e11d352-9fe1-488f-b8b6-e4344ea9c7dc","added_by":"auto","created_at":"2024-12-25 01:22:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":945577,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map of 92 plasma immune biomarkers. Colored vertical bars indicate the biomarkers whose elevation defined the four immunotype groups. Horizontal bars indicate the participants within each immunotype. Blue represents decreased relative protein expression while red represents increased expression.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5504909/v1/bbd4580cb296b9de95386d36.png"},{"id":72300257,"identity":"b3c51607-48dd-4667-9738-5cf4cd034d16","added_by":"auto","created_at":"2024-12-25 01:22:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2309773,"visible":true,"origin":"","legend":"\u003cp\u003eGene Ontology (GO) analysis: A: Immunotype 1; B: Immunotype 2; C: Immunotype 3; D: Immunotype 4\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-5504909/v1/6d0fd00a88ecf20fe4405f00.png"},{"id":72300255,"identity":"bdcc9bba-ebcd-4bdc-a0b7-3fa8ca71993f","added_by":"auto","created_at":"2024-12-25 01:22:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":396130,"visible":true,"origin":"","legend":"\u003cp\u003eANS testing revealed distinct patterns of parasympathetic/vagal, non-cardiovascular sympathetic (i.e. sudomotor), and cardiovascular sympathetic (i.e. adrenergic) deficits, as defined by a CASS sub-score ≥1, in immunotypes 1-4. Diagonal lines indicate prevalence of moderate-severe deficits (CASS sub-score ≥2)\u003c/p\u003e\n\u003cp\u003e* p \u0026lt; 0.001 indicates significant difference between prevalence of cardiovascular sympathetic (i.e. adrenergic) deficits between immunotype 1 and immunotypes 2, 3, and 4.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5504909/v1/845c63135411963e6996c10a.png"},{"id":72300265,"identity":"97a219ca-3fb8-4e16-8496-50a292bb986d","added_by":"auto","created_at":"2024-12-25 01:22:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":284594,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of immune cell type between participants with normal autonomic function and patients with autonomic neuropathy.\u003cstrong\u003e *\u003c/strong\u003e p \u0026lt; 0.01 indicates significant increase in proportion of CD3+ CD8+ and CD8 EMRA (terminally differentiated effector memory T cell) sub-populations of T cells. Autonomic neuropathy is defined as CASS ≥ 3. Normal ANS function defined as CASS ≤ 1.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5504909/v1/343139cc9a67ac5723354feb.png"},{"id":82537475,"identity":"1a4813ec-a069-4112-b14c-82eef63c6e90","added_by":"auto","created_at":"2025-05-12 16:07:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5004978,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5504909/v1/441fbf6e-df93-44cd-a62c-96d3613d9f4c.pdf"},{"id":72300252,"identity":"0a6f41a4-f59a-4bba-9b9a-6cd0cd73be6e","added_by":"auto","created_at":"2024-12-25 01:22:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":70689,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaltables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5504909/v1/885f06e25e491101bdbd4472.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eThe Autonomic Nervous System (ANS)-Immune Network in People Living With HIV\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreclinical studies have demonstrated the importance of the autonomic nervous system (ANS) in regulation of the innate and adaptive immune system. However, our knowledge of the ANS-immune network in humans is limited. Decades of research have established that autonomic neuropathy (AN) is prevalent among people with HIV\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e and HIV-AN is associated with significant morbidity and mortality.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e More recently, AN has been found in patients with chronic inflammatory disorders including Long Covid (LC) syndrome \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)\u003csup\u003e5,6\u003c/sup\u003e, and autoimmune disorders including multiple sclerosis.\u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Despite the significant prevalence of AN in chronic inflammatory disorders, the etiology of the comorbidity is unknown, and a greater understanding of the ANS-immune network is needed.\u003c/p\u003e \u003cp\u003eIn vitro and studies in animals have demonstrated that the extensive peripheral ANS, comprised of sympathetic efferents, parasympathetic (i.e. vagal) efferents, and sensory afferents has the ability to exert targeted, integrated, and rapid modulation of immune signaling through inflammatory reflexes.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Lymphoid organs are highly innervated by sympathetic fibers and the sympathetic neurotransmitter norepinephrine (NE) regulates immune cell production and release of cytokines via α- and β-adrenergic receptors on located on immune cells. At high concentrations, NE released by SNS efferents has an inhibitory effect on immune cells through β-adrenergic receptors,\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e while NE released by the sympathetic adrenal medullary (SAM) axis circulates at lower concentrations, preferentially activating pro-inflammatory, high-affinity α-receptors.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe influence of parasympathetic/vagal activity is primarily anti-inflammatory and often referred to as the cholinergic anti-inflammatory pathway (CAP).\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Pre-clinical studies have established that activation of the CAP results in a reduction of inflammatory cytokine release, including interleukin-6 (IL-6), from splenic macrophages..\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e In vitro studies have demonstrated that the principal neurotransmitter of the parasympathetic nervous system, acetylcholine, released by vagal efferents, has anti-inflammatory effects on immune signaling. In a lipopolysaccharide-stimulated (LPS) human macrophage culture, the application of acetylcholine attenuated the release of pro-inflammatory TNFα, IL-6, and IL-18, but did not influence levels of the anti-inflammatory cytokine IL-10.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur knowledge of ANS regulation of the immune system in humans stems mainly from observational studies focused on parasympathetic/vagal function. Cardiac measurements indicative of lower resting vagal activity are present in patients with inflammatory bowel disease (IBD) as well as rheumatoid arthritis (RA) and neuropsychiatric conditions.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e In addition, vagal nerve stimulation has been associated with reduced levels of pro-inflammatory cytokines in patients with Sjogren\u0026rsquo;s syndrome and Crohn\u0026rsquo;s disease.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e Due to the complexity of the sympathetic nervous system and the methodological challenges associated with its measurement, the influence of sympathetic activity on immune signaling pathways in humans remain largely unexplored. This leaves a significant gap in our understanding of the ANS-immune network, as branches of the ANS do not act independently, but modulate immune signaling through an interconnected network.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eOur study had three goals. As pre-clinical studies have demonstrated the importance of IL-6 in the CAP\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and IL-6\u0026rsquo;s association with increased morbidity and mortality in people with HIV \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, we first investigated the relationship between IL-6 and the parasympathetic/vagal component of baroreflex sensitivity (BRS-V) in people with HIV. We chose to measure BRS-V because this reflex relies on caudal vagal circuitry common to the CAP. \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e Our second goal was to holistically examine the ANS-immune network and determine if the type (i.e. parasympathetic/vagal, cardiovascular sympathetic, and non-cardiovascular sympathetic) and severity of autonomic dysfunction was associated with distinct immune phenotypes identified by unsupervised non-negative matrix factorization (NMF) clustering analyses performed on a panel of 96 biomarkers of inflammation. Finally, as AN is associated with medical morbidity in people with HIV,\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e we sought to compare the burden of non-AIDS-related co-morbidities between immunotypes. Given the availability of medications and devices that modulate both the ANS and the immune system, we hope a greater understanding of the ANS-immune network will rapidly translate into therapeutic studies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e2.1 Study Design and Patient Population.\u003c/strong\u003e This is a cross-sectional observational study. Participants were recruited from a primary care clinic network within the Mount Sinai Health System in New York City which provides primary care to more than 10,000 people living with HIV. Eligible participants were identified by pre-screening clinic providers\u0026rsquo; schedules and requesting approval from providers prior to contacting potential participants. Included participants were at least 18 years of age with a history of well-controlled HIV infection (plasma RNA load of \u0026le;\u0026thinsp;100 copies/ml for 3 months prior to enrollment) who were on a stable combination of antiretroviral treatment (CART) for at least 3 months. Patients with another diagnosis known to cause autonomic dysfunction (e.g., diabetes) not able to complete required autonomic testing (e.g., participant must be able to stand) or taking substances that impact the autonomic nervous system (e.g. urine drug testing positive for stimulants) were excluded. All procedures were performed in accordance with a protocol approved by the Institutional Review Board of the Icahn School of Medicine at Mount Sinai (ISMMS) and all participants provided written informed consent.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.2 Autonomic testing procedures\u003c/strong\u003e: Autonomic function tests (AFTs) are a standard battery of non-invasive tests (WRMed) which include sudomotor testing (QSWEAT), heart rate response to deep breathing, Valsalva maneuver (VM), and tilt table testing. The QSWEAT is performed by placing a capsule containing acetyl choline (ACh) on the skin in four standardized locations (forearm, proximal leg, distal leg, and foot). The capsule is attached to an automated system which delivers a small continuous electrical stimulus to the capsule causing iontophoresis of ACh into the skin, which triggers a reflexive sweat response collected by the capsule. The evoked sweat volume is measured and compared to standardized values. A non-invasive continuous beat-to-beat blood pressure (BP) monitoring device is attached to the participant\u0026rsquo;s finger and a 3-lead surface electrocardiogram and respiratory monitor are attached to the chest. BP, heart rate (HR), and respirations are recorded during the VM (forced exhalation to a pressure of 40 mmHg for 15 seconds), standardized paced deep breathing (HRDB), and a 10-minute head-up tilt test.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.3 Calculation of autonomic indices\u003c/strong\u003e: The above-described procedures are used to calculate the Composite Autonomic Severity Score (CASS), which is an age- and sex-adjusted summary score reflecting overall autonomic function and is the sum of three sub-scores. The sudomotor (i.e. peripheral, non-cardiovascular sympathetic) sub-score uses data from the QSWEAT, the parasympathetic/vagal sub-score is based on changes in HR during deep breathing and VM, and the adrenergic (i.e., cardiovascular sympathetic) sub-score is based on BP changes during VM and tilt table testing. Given the medical complexity of our patient population, our lab uses the stringent threshold of a total CASS\u0026thinsp;\u0026ge;\u0026thinsp;3 to define AN; a sub-score of 1 defines mild dysfunction and a sub-score\u0026thinsp;\u0026ge;\u0026thinsp;2 identifies moderate to severe dysfunction.\u003csup\u003e\u003cspan\u003e24\u003c/span\u003e\u003c/sup\u003e\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eBaroreflex sensitivity (BRS) was calculated as previously described.\u003csup\u003e\u003cspan\u003e25\u003c/span\u003e\u003c/sup\u003e Briefly, VM data is visually inspected by a trained, blinded technician. BRS-V is a measure of the compensatory cardiac response to a decrease in BP evoked during the forced expiration against a closed glottis and is calculated by dividing the change in RR interval during phase 2E of the VM by the change in systolic blood pressure. It is a continuous measurement expressed as milliseconds/mmHg. BRS-A is expressed in mmHg/second, and it is calculated by dividing the change in systolic blood pressure during phase 3 by the time required for SBP to recover following the release of VM.\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e2.4 Medical history and patient-reported outcome measures\u003c/strong\u003e: Medical history and concomitant medications were obtained through participant interview and review of the electronic health record (EHR). To control potentially confounding influences of medications and comorbidities, several indices were included in multivariable analyses. First, as acetylcholine is a main neurotransmitter of the ANS, an anticholinergic burden (ACB) medication score was determined for all participants. Second, a Charlson Comorbidity Index was calculated (modified to exclude HIV/AIDS).\u003csup\u003e26\u0026ndash;28\u003c/sup\u003e Finally, as there is significant overlap between the limbic neural circuitry that underlies emotion regulation and rostral pathways of the central autonomic network (CAN), participants completed the Hospital Anxiety and Depression Scale (HADS)\u003csup\u003e\u003cspan\u003e29\u003c/span\u003e\u003c/sup\u003e and the Perceived Stress Scale-14 (PSS-14).\u003csup\u003e\u003cspan\u003e30\u003c/span\u003e\u003c/sup\u003e\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.5 Biospecimen Collection and Proteomics Analysis\u003c/strong\u003e: Whole blood was collected in EDTA tubes followed by isolation of peripheral blood mononuclear cell (PBMC) and plasma, as described previously.\u003csup\u003e\u003cspan\u003e31\u003c/span\u003e\u003c/sup\u003e Serum was obtained concomitantly from serum-separator tubes. At our institution\u0026rsquo;s Human Immune Monitoring Center (HIMC), a shared core research facility, plasma IL-6 was measured as part of the Target 96 Inflammation Panel on the Olink proteomics platform (Uppsala, Sweden) as previously published.\u003csup\u003e\u003cspan\u003e31\u003c/span\u003e\u003c/sup\u003e\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.6 Cytometry by time of flight (CyTOF)\u003c/strong\u003e: CyTOF was performed on PBMC samples from participants with AN (CASS\u0026thinsp;\u0026ge;\u0026thinsp;3) and without (CASS\u0026thinsp;\u0026le;\u0026thinsp;1) selected purposively to maximize the difference in CASS between the groups while balancing sex and age (N\u0026thinsp;=\u0026thinsp;5 per group) and without knowledge of cytokine profiles. Briefly, cell counts were performed on the Nexcelom Cellaca Automated Cell Counter (Nexcelom Biosciences) and cell viability was measured using Acridine Orange/Propidium Iodide viability staining reagent (Nexcelom). After washing cells in Cell Staining Buffer (CSB) (Fluidigm) Fc receptor blocking (Biolegend) and Rhodium-103 viability staining (Fluidigm) were performed simultaneously with surface markers for 30 minutes at room temperature. Cells were subsequently washed twice in CSB and then palladium barcoding was performed on each sample using the Cell-ID 20-Plex Pd Barcoding Kit (Fluidigm) following manufacturer\u0026rsquo;s instructions and pooled together. The pooled sample was fixed with 2.4% PFA (Electron Microscopy Sciences) followed by labeling with 125nM Iridium-193 (Fluidigm) and 2nM Osmium tetroxide (EMS) for 30 minutes at room temperature. Immediately prior to data acquisition, samples were resuspended at a concentration of 1\u0026nbsp;million cells per ml in Cell Acquisition Solution containing a 1:20 dilution of EQ Normalization beads (Fluidigm). The samples were acquired on a Helios Mass Cytometer (Fluidigm) equipped with a wide-bore sample injector at an event rate of \u0026lt;\u0026thinsp;400 events per second. Prior to analysis, routine data normalization (Fluidigm software) and sample demultiplexing were undertaken.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.7 Statistical analysis\u003c/strong\u003e: All data were stored in REDCap. Descriptive statistics including frequencies, percents and medians with interquartile range were calculated as appropriate. Spearman rank correlation was performed to investigate the relationship between IL-6 and BRS-V. While CASS normative scoring is adjusted for age and sex, BRS is not. Therefore, multivariate logistic regression adjusting for age and sex was performed for analyses involving BRS. Kruskal-Wallis or Chi-square compared continuous and categorical variables between the four immunotypes, respectively. Bonferroni correction was applied to analyses involving multiple comparisons.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003eImmunotypes were defined using the R package for nonnegative matrix factorization (NMF) as previously described.\u003csup\u003e\u003cspan\u003e32\u003c/span\u003e\u003c/sup\u003e Normalized protein expression of Olink inflammatory markers were compared between immunotypes. STRINGdb R package v2.16.4 was used for gene ontology (GO) enrichment analysis to elucidate key biological processes associated with immunotypes. The significance of GO terms was determined based on P-value (\u0026lt;\u0026thinsp;0.001) and FDR (\u0026lt;\u0026thinsp;0.05). To account for multiple testing and reduce the likelihood of false positives, we applied the Benjamini-Hochberg test for corrections. All analyses were conducted using SPSS version 28 and R 4.3.0.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Study population demographics, clinical characteristics, and autonomic function\u003c/h2\u003e \u003cp\u003eParticipant demographics and medical characteristics are summarized in Table\u0026nbsp;1. Participants (N\u0026thinsp;=\u0026thinsp;79) had an average age of 51.6 years (range: 25\u0026ndash;73 years of age) and approximately three quarters were male. The majority had long-standing HIV with a self-reported mean of 22 years. African American was the most common race/ethnicity (48%), while 17% of participants were Hispanic/LatinX, 17% were non-Hispanic/LatinX white and 17% identified as other. With regard to medication, the mean ACB score for participants was 0.3 (SD\u0026thinsp;=\u0026thinsp;0.8) with a range of 0 to 4. Regarding medical comorbidities, the median Charlson was 1.0 (IQR 0\u0026ndash;1), which aligns with previously reported values in people living with HIV.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e Hypertension and hyperlipidemia were the most common comorbidities in our population (38.0% and 31.7% respectively; Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e76/79 participants completed autonomic nervous system testing. The total CASS ranged from 0\u0026ndash;6 with a median score of 2.0. AN was common (44.7%) in our study population. Examination of CASS sub-scores showed that 42% of all participants had adrenergic (i.e., cardiovascular sympathetic) sub-score abnormalities, 57% had sudomotor (i.e. non-cardiovascular sympathetic) abnormalities, and 49% had cardiovagal (i.e., vagal/parasympathetic) dysfunction. In patients with AN, dysfunction across multiple domains of ANS function was common, with dysfunction in two domains present in 58.9% of participants and 29.4% demonstrating dysfunction in all three domains.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Increased IL-6 In Patients with Parasympathetic/Vagal Dysfunction\u003c/h2\u003e \u003cp\u003eWe first explored the relationship between IL-6 and parasympathetic/vagal function. Spearman rank correlation revealed a significant univariate correlation between IL-6 and BRS-V in the expected direction with reduced vagal function correlating to higher IL-6 (Spearman\u0026rsquo;s rho = -0.352, p\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This was confirmed with multivariate, stepwise, linear regression with IL-6 as the outcome variable and BRS-V, age, ACB score, CD4+, sex, Charlson score, and diagnoses of anxiety and depression as the predictors; the final model retained BRS-V (p\u0026thinsp;=\u0026thinsp;0.012) and age (p\u0026thinsp;=\u0026thinsp;0.028).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Unsupervised Clustering Identified a Pro-inflammatory Immunotype with Older Participants, and a High Prevalence of AN and Burden of Co-morbid Illness\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we assessed the profile of inflammatory proteins using unbiased, non-negative matrix factorization (NMF) and found four clusters or immunotypes, with distinct differentially expressed protein expression profiles, as demonstrated in the resulting heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These immunotypes differed with respect to prevalence and severity of AN, age, as well as burden of co-morbid illnesses (Tables\u0026nbsp;1 and 2). Of note, sex, CD4\u0026thinsp;+\u0026thinsp;count, HADS scores, ACB score, and PSS-14 scores did not differ between the four immunotypes (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImmunotype 1\u003c/span\u003e was defined by an upregulation of pro-inflammatory biomarkers including interleukins (IL-6, IL-17), and members of the TNF families (IL-18, TNF-β, TNFRSF9, OPG) which are regulated by the CAP. Immunotype 1 also had increased levels of interleukins involved in the expansion and activation of CD8\u0026thinsp;+\u0026thinsp;T- cells, including IL-12B, IL-17c, and IL-10RB, and decreased levels of proteins that act to inhibit T-cell expansion including CASP-8.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e (See Supplemental Table\u0026nbsp;1). GO analyses demonstrated an enrichment in proteins involved in T-helper 1 cell cytokine production, as well as the migration and chemotaxis of several immune cell subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImmunotype 1 was six years older than the study population overall (60.5 years versus 53.5 years) and had a higher median Charlson Comorbidity Index compared to other immunotypes. The most prevalent medical comorbidities were hypertension (47.6%) and hyperlipidemia (42.9%). Immunotype 1 also had the highest prevalence of AN (70%). In multivariate logistic regression participants with AN were almost five times more likely to be immunotype 1 than immunotypes 2\u0026ndash;4 (aOR\u0026thinsp;=\u0026thinsp;4.7, p\u0026thinsp;=\u0026thinsp;0.017) after adjusting for age, sex, ABC score, Charlson Index, and CD4\u0026thinsp;+\u0026thinsp;count. All participants with moderate to severe AN (CASS\u0026thinsp;\u0026gt;\u0026thinsp;4) were in immunotype 1 and no participants with a CASS of zero were in immunotype 1. Cardiovascular sympathetic nervous system deficits defined this immunotype as they were present in three-quarters of participants in immunotype 1, compared to just 20\u0026ndash;30% in other immunotypes. (Table\u0026nbsp;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; p\u0026thinsp;=\u0026thinsp;0.001)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImmunotype 2\u003c/span\u003e also demonstrated a pro-inflammatory profile. Examination of normalized protein expressed showed that many of the immunotype 1 defining proteins, including IL-18 and TNF-β, were upregulated in immunotype 2, though to a lesser extent (Supplemental Table\u0026nbsp;1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, while NMF analyses identified the pro-inflammatory vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), IL-12, and Fms-related tyrosine kinase 3 ligand (FLT3LG) as immunotype 2 defining proteins, these proteins were also upregulated in immunotype 1, though to a lesser extent. Immunotype 2 also demonstrated an increased level of \u003cem\u003eanti-\u003c/em\u003einflammatory cytokines and proteins indicative of cell apoptosis and the negative regulation of cytokine production (e.g. IL-10, TNFSF10) which were not upregulated in immunotype 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results aligned with pathways identified in GO enrichment analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eImmunotype 2 was significantly younger than immunotype 1 (47.0 versus 60.5 years) and had a significantly lower Charlson Comorbidity Index than immunotype 1 (0.5 versus 2.0). Immunotype 2 also had the shortest HIV disease duration, though this was not a statistically different result. Immunotype 2 had a lower prevalence of AN (44.8%) than immunotype 1, which was due to a reduced prevalence of adrenergic deficits (20% versus 75%).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImmunotype 3\u003c/span\u003e had a different immune signaling profile compared to immunotypes 1 and 2. The pro-inflammatory proteins elevated in immunotypes 1 and 2 were not elevated in immunotype 3 (Supplemental Table\u0026nbsp;1) and similar to immunotype 2, GO enrichment analysis demonstrated upregulation of proteins involved in the \u003cem\u003enegative\u003c/em\u003e regulation of cytokine production and differentiation of immune cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Defining proteins identified by NMF included the glial derived protein family (GDNF and NTN), neurotrophin 3 (NTF3) and IL-4, a cytokine involved in the differentiation of TH cells into TH2 cells.\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDemographically, the age and comorbidity burden of immunotype 3 did not differ from the study population average. However, immunotype 3 had significantly more African Americans than other immunotypes (87.5%).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImmunotype 4\u003c/span\u003e also had a lower inflammatory profile compared to immunotypes 1 and 2, though there was significant upregulation of several pro-inflammatory proteins related to immune cell chemotaxis including CXCL11 and monocyte chemoattractant protein-1 (MCP-1), which distinguished it from immunotype 3. (Supplemental table 1). GO analysis identified enriched expression of pathways involved in intracellular signaling and positive regulation of immunoglobulin secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eImmunotype 4 had the highest prevalence of solid tumors, HSV, and asthma compared to other immunotypes, although this was not statistically significant. Approximately one-third of participants in immunotype 4 had AN (36.4%) but, like immunotype 3, the majority of autonomic dysfunction was mild (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.4 CyTOF:\u003c/h2\u003e \u003cp\u003eTo gain a greater understanding of how AN alters the immune profile, we performed a cellular profile analysis using high-parameter CyTOF, comparing the cellular composition between patients diagnosed with and without AN. As described in the methods, participants were selected purposefully to maximize the difference in CASS, and without knowledge of their cytokine profiles. Interestingly, we found that all patients in the AN group belonged to immunotype 1. CyTOF results showed an increased abundance of CD8\u0026thinsp;+\u0026thinsp;T-cells in patients with AN compared to those without AN, which was particularly pronounced for the terminally differentiated effector memory subtype of the CD8\u0026thinsp;+\u0026thinsp;T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe utilized comprehensive ANS testing and a broad-scale proteomic approach to assess the ANS-immune network in 79 people living with HIV. We found autonomic neuropathy (AN) was prevalent (44.7%) and predicted a pro-inflammatory phenotype characterized by elevations in type-1 cytokines, a predominance of CD8\u0026thinsp;+\u0026thinsp;T-cells, and a higher burden of co-morbid illness in people living with HIV. These findings provide novel evidence for the clinical significance of parasympathetic/vagal and sympathetic pathways in the regulation of immune signaling.\u003c/p\u003e \u003cp\u003eThis is the first study to demonstrate that diminished activity of caudal parasympathetic/vagal circuitry, measured by vagal baroreflex sensitivity, predicted elevated IL-6 in people with HIV. IL-6 plays a pivotal role in vascular inflammation, endothelial dysfunction, and adverse cardiovascular events.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Yet, despite pre-clinical studies demonstrating IL-6\u0026rsquo;s regulation by the CAP, our understanding of this pathway in humans is limited. The majority of studies examining the relationship between parasympathetic/vagal activity and systemic inflammation use resting heart rate variability (HRV) measurements.\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e However, resting HRV is associated with the activity of prefrontal-amygdala pathways \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003eand is decreased in stress-related mood disorders.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Our own lab found reduced resting HRV in patients with normal peripheral vagal pathways.\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e We found only one study that used a reflexive measure of vagal activity to assess the relationship between plasma IL-6 and caudal parasympathetic/vagal circuitry. In young patients with type I diabetes without AN, lower HRDB correlated to higher levels of plasma IL-6.\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e Thus, our results provide novel support for the relevance of the CAP in patients with chronic inflammatory AN.\u003c/p\u003e \u003cp\u003eUnsupervised NMF clustering analysis revealed distinct immunotypes in people with HIV, supporting previous work that has demonstrated low and high inflammatory subgroups in people with HIV.\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e Immunotype 1 was significantly older and had a higher burden of comorbid illness compared to immunotype 2. While both immunotypes 1 and 2 demonstrated upregulation of pro-inflammatory proteins associated with Type 1 T-cell expansion, immunotype 2 also had an enrichment of proteins involved in the \u003cem\u003enegative\u003c/em\u003e regulation of inflammation and apoptosis, which was supported by GO analysis. The upregulation of these inhibitory proteins distinguished immunotype 2 from immunotype 1. The enrichment of CD8\u0026thinsp;+\u0026thinsp;T-cells in patients with AN supports the proteomics analyses showing an upregulation of plasma immune biomarkers associated with proliferation of Type 1 T-cells in immunotype 1. The association of HIV-AN with a greater abundance of CD8\u0026thinsp;+\u0026thinsp;T-cells is particularly relevant given that low CD4/CD8 ratio has been implicated in multiple poor outcomes in people with HIV including neurocognitive disorders, malignancy, and cardiovascular disease.\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSurprisingly, the pro-inflammatory immunotype 1 did not display a greater prevalence or severity of parasympathetic/vagal dysfunction. Instead, pathology in the sympathetic nervous system distinguished immunotype 1 from the younger and healthier immunotype 2. The increased prevalence of cardiovascular sympathetic deficits was particularly striking (75% in immunotype 1 versus 20% in immunotype 2). Non-cardiovascular sympathetic deficits, as measured by the sudomotor sub-score, were also more common in immunotype 1, indicating widespread sympathetic dysfunction. Pre-clinical studies provide an anatomic and physiologic basis of the anti-inflammatory impact of the sympathetic nervous system.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e In vitro studies have demonstrated that local release of NE from sympathetic efferents inhibits the production and release of pro-inflammatory cytokines through actions at β-adrenergic receptors located on the surface of immune cells.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e While we could not directly assess the sympathetic efferents innervating organs of the immune system, in more advanced HIV-AN with evidence of both cardiovascular and non-cardiovascular sympathetic impairment (as immunotype 1 displayed), it would be unlikely that they would be selectively spared.\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e Moreover, loss of sympathetic innervation of lymph nodes has been demonstrated in Simian Immunodeficiency Virus (SIV) SIV models.\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e Future studies should examine if organ-specific deficits in post-ganglionic sympathetic efferent activity are present in people living with HIV.\u003c/p\u003e \u003cp\u003eThe significant age difference between immunotype 1 (median 60.5 years) and 2 (median 47.5 years) warrants discussion. Determining the influence of aging on HIV-AN progression may be confounded by the common correlation between age and duration of HIV disease and the fact that younger people with HIV did not experience the early HIV epidemic (i.e., lack of treatment followed by neurotoxic treatments). Interestingly, disease duration did not significantly differ between immunotypes 1 and 2 and suggests that aging may contribute to deficits in cardiovascular sympathetic activity that characterize immunotype 1. We hypothesize that with time, the autonomic, immune and clinical characteristics of immunotype 2 may evolve to resemble those of immunotype 1. Clinical and pre-clinical studies indicate aging comprises peripheral nerve structure and function through mechanisms that involve inflammation, and thus may be accelerated in the context of the pathologic inflammation of chronic HIV.\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e Unmyelinated, type C fibers of the sympathetic nervous system may be more vulnerable to aging than type A and B vagal efferent projections.\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e Longitudinal research examining the progression of HIV-AN and its influence on the immune network is needed to explore this hypothesis.\u003c/p\u003e \u003cp\u003eThe diminished prevalence of AN in immunotype 3 may have contributed to its lower inflammatory profile. However, it is important to note that a significantly greater proportion of immunotype 3 identified as African American (87.5%) and while isolating the biological influence of race on the immune system is complicated by cultural and psychosocial influences, there is evidence for race-based differences in immune signaling which may confound conclusions regarding the ANS\u0026rsquo;s influence on immune signaling.\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e Similarly, immunotype 4 had a higher prevalence of solid tumors, HSV, and asthma, and thus, AN is unlikely to be the primary influence on immune signaling.\u003c/p\u003e \u003cp\u003eThere are certain limitations to our study. First, the cross-sectional design and a small sample size prohibit establishing mechanistic causation and larger, longitudinal studies are needed. However, this work does provide important support for a pre-clinical literature that demonstrates direct modulation of the immune system by the ANS. Second, while female representation in our study aligns with HIV disease prevalence, the lower enrollment makes it difficult to examine the likely influence of sex differences on ANS-immune function. Finally, additional tests of autonomic dysfunction such as catecholamine plasma levels in response to standing or intraepidermal nerve fiber density on skin biopsy were not obtained. Therefore, to acknowledge this limitation, we chose a higher CASS threshold to identify autonomic neuropathy.\u003c/p\u003e \u003cp\u003eIn conclusion, our results provide important evidence that the cholinergic anti-inflammatory pathway (CAP), established by pre-clinical studies, may be relevant to the pathology of chronic inflammatory disorders. In addition, these data demonstrate the importance of the sympathetic nervous system in the development of a pro-inflammatory immune signature associated with a higher burden of co-morbid disease and suggest that longitudinal examination of the time course of HIV-AN progression may provide important insight regarding the influence of aging on this process. Finally, our findings illustrate that a comprehensive evaluation of the ANS-immune network provides a greater understanding of core mechanisms that lead to immune system dysregulation associated with increased morbidity and mortality in people with HIV and provide a rationale for future research to develop therapeutic strategies focused on modulation of the sympathetic and parasympathetic/vagal nervous system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAcknowledgments:\u003c/u\u003e\u003c/strong\u003e This work was supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK; R01DK122853) and the National Institute of Health (NIH) Helping to End Addition Long Term (HEAL: K12NS130673). This work was also supported in part through the computational and data resources and staff expertise provided by Scientific Computing and Data at the Icahn School of Medicine at Mount Sinai and supported by the Clinical and Translational Science Awards (CTSA) grant UL1TR004419 from the National Center for Advancing Translational Sciences.\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest relevant to this work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRobinson-Papp J, Sharma S, Simpson DM, Morgello S. Autonomic dysfunction is common in HIV and associated with distal symmetric polyneuropathy. \u003cem\u003eJ Neurovirol\u003c/em\u003e. Apr 2013;19(2):172-80. doi:10.1007/s13365-013-0160-3\u003c/li\u003e\n\u003cli\u003eKwon PM, Lawrence S, Figueroa A, Robinson-Papp J. 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A systematic review and meta-analytic confirmatory factor analysis of the perceived stress scale (PSS-10 and PSS-14). \u003cem\u003eStress Health\u003c/em\u003e. Feb 2024;40(1):e3285. doi:10.1002/smi.3285\u003c/li\u003e\n\u003cli\u003eAssarsson E, Lundberg M, Holmquist G, et al. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability. \u003cem\u003ePLoS One\u003c/em\u003e. 2014;9(4):e95192. doi:10.1371/journal.pone.0095192\u003c/li\u003e\n\u003cli\u003eGaujoux R, Seoighe C. A flexible R package for nonnegative matrix factorization. \u003cem\u003eBMC Bioinformatics\u003c/em\u003e. Jul 2 2010;11:367. doi:10.1186/1471-2105-11-367\u003c/li\u003e\n\u003cli\u003eRamirez HC, Monroe AK, Byrne M, O\u0026apos;Connor LF. Examining the Association Between a Modified Quan-Charlson Comorbidity Index and HIV Viral Suppression: A Cross-Sectional Analysis of DC Cohort Participants. \u003cem\u003eAIDS Res Hum Retroviruses\u003c/em\u003e. Dec 2023;39(12):662-670. doi:10.1089/AID.2022.0186\u003c/li\u003e\n\u003cli\u003eEmmerich J, Mumm JB, Chan IH, et al. IL-10 directly activates and expands tumor-resident CD8(+) T cells without de novo infiltration from secondary lymphoid organs. \u003cem\u003eCancer Res\u003c/em\u003e. Jul 15 2012;72(14):3570-81. doi:10.1158/0008-5472.CAN-12-0721\u003c/li\u003e\n\u003cli\u003eLi-Weber M, Krammer PH. Regulation of IL4 gene expression by T cells and therapeutic perspectives. \u003cem\u003eNat Rev Immunol\u003c/em\u003e. Jul 2003;3(7):534-43. doi:10.1038/nri1128\u003c/li\u003e\n\u003cli\u003ePatterson CC, Smith AE, Yarnell JW, Rumley A, Ben-Shlomo Y, Lowe GD. The associations of interleukin-6 (IL-6) and downstream inflammatory markers with risk of cardiovascular disease: the Caerphilly Study. \u003cem\u003eAtherosclerosis\u003c/em\u003e. Apr 2010;209(2):551-7. doi:10.1016/j.atherosclerosis.2009.09.030\u003c/li\u003e\n\u003cli\u003eBerger M, Marz W, Niessner A, et al. IL-6 and hsCRP predict cardiovascular mortality in patients with heart failure with preserved ejection fraction. \u003cem\u003eESC Heart Fail\u003c/em\u003e. Jul 14 2024;doi:10.1002/ehf2.14959\u003c/li\u003e\n\u003cli\u003eAronson D, Mittleman MA, Burger AJ. Interleukin-6 levels are inversely correlated with heart rate variability in patients with decompensated heart failure. \u003cem\u003eJ Cardiovasc Electrophysiol\u003c/em\u003e. Mar 2001;12(3):294-300. doi:10.1046/j.1540-8167.2001.00294.x\u003c/li\u003e\n\u003cli\u003eWei L, Chen H, Wu G-R. Structural Covariance of the Prefrontal-Amygdala Pathways Associated with Heart Rate Variability. \u003cem\u003eFrontiers in Human Neuroscience\u003c/em\u003e. 2018;12doi:10.3389/fnhum.2018.00002\u003c/li\u003e\n\u003cli\u003eThayer JF, Lane RD. Claude Bernard and the heart-brain connection: further elaboration of a model of neurovisceral integration. \u003cem\u003eNeurosci Biobehav Rev\u003c/em\u003e. Feb 2009;33(2):81-8. doi:10.1016/j.neubiorev.2008.08.004\u003c/li\u003e\n\u003cli\u003eKwon PM, Lawrence S, Mueller BR, Thayer JF, Benn EKT, Robinson-Papp J. Interpreting resting heart rate variability in complex populations: the role of autonomic reflexes and comorbidities. \u003cem\u003eClin Auton Res\u003c/em\u003e. Jun 2022;32(3):175-184. doi:10.1007/s10286-022-00865-2\u003c/li\u003e\n\u003cli\u003eGonzalez-Clemente JM, Vilardell C, Broch M, et al. Lower heart rate variability is associated with higher plasma concentrations of IL-6 in type 1 diabetes. \u003cem\u003eEur J Endocrinol\u003c/em\u003e. Jul 2007;157(1):31-8. doi:10.1530/EJE-07-0090\u003c/li\u003e\n\u003cli\u003eVadaq N, van de Wijer L, van Eekeren LE, et al. Targeted plasma proteomics reveals upregulation of distinct inflammatory pathways in people living with HIV. \u003cem\u003eiScience\u003c/em\u003e. Oct 21 2022;25(10):105089. doi:10.1016/j.isci.2022.105089\u003c/li\u003e\n\u003cli\u003eWolday D, Kebede Y, Legesse D, et al. Role of CD4/CD8 ratio on the incidence of tuberculosis in HIV-infected patients on antiretroviral therapy followed up for more than a decade. \u003cem\u003ePLoS One\u003c/em\u003e. 2020;15(5):e0233049. doi:10.1371/journal.pone.0233049\u003c/li\u003e\n\u003cli\u003eLow PA. Evaluation of sudomotor function. \u003cem\u003eClin Neurophysiol\u003c/em\u003e. Jul 2004;115(7):1506-13. doi:10.1016/j.clinph.2004.01.023\u003c/li\u003e\n\u003cli\u003eSloan EK, Nguyen CT, Cox BF, Tarara RP, Capitanio JP, Cole SW. SIV infection decreases sympathetic innervation of primate lymph nodes: the role of neurotrophins. \u003cem\u003eBrain Behav Immun\u003c/em\u003e. Feb 2008;22(2):185-94. doi:10.1016/j.bbi.2007.07.008\u003c/li\u003e\n\u003cli\u003eShibuta Y, Nodera H, Mori A, Okita T, Kaji R. Peripheral nerve excitability measures at different target levels: the effects of aging and diabetic neuropathy. \u003cem\u003eJ Clin Neurophysiol\u003c/em\u003e. Oct 2010;27(5):350-7. doi:10.1097/WNP.0b013e3181f387ab\u003c/li\u003e\n\u003cli\u003eHappe M, Samuvel DJ, Ohtola JA, Korte JE, Westerink MAJ. Race-related differences in functional antibody response to pneumococcal vaccination in HIV-infected individuals. \u003cem\u003eVaccine\u003c/em\u003e. Mar 14 2019;37(12):1622-1629. doi:10.1016/j.vaccine.2019.01.084\u003c/li\u003e\n\u003cli\u003eYuan H, Silberstein SD. Vagus Nerve and Vagus Nerve Stimulation, a Comprehensive Review: Part I. \u003cem\u003eHeadache\u003c/em\u003e. Jan 2016;56(1):71-8. doi:10.1111/head.12647\u003c/li\u003e\n\u003cli\u003eSchindler SE, Cruchaga C, Joseph A, et al. African Americans Have Differences in CSF Soluble TREM2 and Associated Genetic Variants. \u003cem\u003eNeurol Genet\u003c/em\u003e. Apr 2021;7(2):e571. doi:10.1212/NXG.0000000000000571\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 619px;\"\u003e\n \u003cp\u003eTable 1: \u0026nbsp; \u0026nbsp; Participant Demographics\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003eOverall\u003c/p\u003e\n \u003cp\u003eN = 79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003eImmunotype 1\u003c/p\u003e\n \u003cp\u003eN = 21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003eImmunotype 2\u003c/p\u003e\n \u003cp\u003eN = 29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003eImmunotype 3\u003c/p\u003e\n \u003cp\u003eN = 16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eImmunotype 4\u003c/p\u003e\n \u003cp\u003eN = 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eAge, years*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e53.5 (42.0, 61.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e60.5\u003c/p\u003e\n \u003cp\u003e(52.8, 63.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e47.0 (18.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e52.5 (21.25)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e54.0 (27.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.043\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eSex, male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e58 (73.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e14 (66.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e22 (75.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e11 (68.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e8 (72.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.778\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eRace ethnicity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eAfrican American\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e38(48.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e6 (29.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e15 (51.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e14 (87.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2 (18.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eHispanic/LatinX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e14(17.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e7 (33.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e5 (17.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1 (9.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eWhite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e13(16.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e4 (19.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e4 (13.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1 (6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e4 (36.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e14(17.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e3 (14.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e5 (17.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1 (6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3 (27.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eLatest CD4+ count (cells/mm\u003csup\u003e3\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e578.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(463.5,815.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e584.5\u003c/p\u003e\n \u003cp\u003e(244.0,837.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e640.5\u003c/p\u003e\n \u003cp\u003e(496.3, 851.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e547.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(437.3, 855.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e521.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(350.0, 758.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.496\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eTime since HIV diagnosis, years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003cp\u003e(15.0, 32.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e24.8\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(19.4, 30.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e21.1\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(17.3, 25.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e24.3\u003c/p\u003e\n \u003cp\u003e(19.2, 29.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003cp\u003e(19.4, 30.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.521\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eHADS Anxiety\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e7.1\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(2.6, 11.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e7.6\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(5.1,10.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e6.6\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(5.1, 8.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e7.8\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(5.2,10.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e7.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(3.7, 10.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.818\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eHADS Depression\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e4.6\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(0.81, 8.39)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e5.4\u003c/p\u003e\n \u003cp\u003e(3.5,7.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e4.1\u003c/p\u003e\n \u003cp\u003e(2.8,5.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5.2\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(3.2,7.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3.8\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(0.9, 6.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.541\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003ePSS-14 Score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e16.5\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(9.0, 24.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e16.3\u003c/p\u003e\n \u003cp\u003e(12.5, 20.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e14.9\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(12.4, 17.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e16.9\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(12.3, 21.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e15.8\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(9.4, 22.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.391\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eCCI Score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e1.0 (0.0, 2.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e2.0 (1.0, 5.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.5 (0.0, 2.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1.0 (0.0, 2.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1.0 (0.0, 6.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eACB Score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e0.3 (-0.5,1.1)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e0.7 (0.1 1.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e0.2 (0.0, 0.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0.2 (0.0, 0.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.2 (0.0, 0.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.798\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"9\" valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003eComorbidities\u003c/p\u003e\n \u003cp\u003eHypertension\u003c/p\u003e\n \u003cp\u003eHyperlipidemia\u003c/p\u003e\n \u003cp\u003eCAD\u003c/p\u003e\n \u003cp\u003eSyphilis history\u003c/p\u003e\n \u003cp\u003eHSV\u003c/p\u003e\n \u003cp\u003eAsthma\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Obesity\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eOsteoarthritis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e30 (38.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e10 (47.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e13 (44.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5 (31.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2 (18.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.329\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e25 (31.65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e9 (42.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e8 (27.6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e5 (31.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3 (27.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.570\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e3 (3.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e2 (9.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e1 (3.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1 (6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.387\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e19 (24.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e3 (14.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e11 (37.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3 (27.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.225\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e9 (11.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e4 (19.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e1 (3.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1 (6.3)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3 (27.3)\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.086\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e14 (17.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e4 (19.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e5 (17.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e2 (12.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3 (27.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.819\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e5 (6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e3 (14.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e1 (3.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1 (6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.308\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e7 (8.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e4 (19.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e2 (6.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e0 (0.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1 (9.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.209\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 114px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Solid tumor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e9 (11.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 86px;\"\u003e\n \u003cp\u003e3 (14.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e2 (6.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 90px;\"\u003e\n \u003cp\u003e1 (6.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3 (27.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 55px;\"\u003e\n \u003cp\u003e0.262\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 619px;\"\u003e\n \u003cp\u003eUnless otherwise stated, data are summarized as mean (95% confidence interval) or N (% of column).\u003c/p\u003e\n \u003cp\u003e*, median (interquartile range).\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eEach subscript letter denotes a subset of immunotype categories whose column proportions do not significantly differ from each other at the 0.05 level.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 619px;\"\u003e\n \u003cp\u003eAbbreviations: CD4: Cluster of Differentiation 4; HADS: Hospital Anxiety and Depression Scale; PSS-14: Perceived Stress Scale; ACB: Anticholinergic Burden; CAD: Coronary Artery Disease; HSV: Herpes Simplex Virus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 624px;\"\u003e\n \u003cp\u003eTable 2: ANS activity of immunotypes\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eOverall\u003c/p\u003e\n \u003cp\u003eN = 76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003eImmunotype 1\u003c/p\u003e\n \u003cp\u003eN = 20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003eImmunotype 2\u003c/p\u003e\n \u003cp\u003eN = 29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eImmunotype 3\u003c/p\u003e\n \u003cp\u003eN = 16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eImmunotype 4\u003c/p\u003e\n \u003cp\u003eN = 11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eTotal CASS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 83px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003cp\u003e[1.0,3.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.5\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[2.0, 4.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e2.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.0, 3.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003cp\u003e[1.0, 2.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e2.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.0, 3.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.005\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026lt; 0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eAutonomic neuropathy\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(CASS \u0026ge; 3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83px;\"\u003e\n \u003cp\u003e34 (44.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e14 (70.0)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e13 (44.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3 (18.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e4 (36.4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.020\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026lt; 0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 139px;\"\u003e\n \u003cp\u003eSudomotor CASS\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83px;\"\u003e\n \u003cp\u003e1.0 [0.0,2.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e2.0 [0.0, 4.5]\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.0 [0.0,2.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.0 [0.0, 2.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0.0 [0.0, 2.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.265\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e0.048\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 139px;\"\u003e\n \u003cp\u003eAdrenergic CASS \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83px;\"\u003e\n \u003cp\u003e0.0 [0.0,1.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e1.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.3,1.8]\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003cp\u003e[ 0.0, 0.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.0, 1.0]\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003cp\u003e[0.0, 1.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e\u0026lt; 0.001\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e\u0026lt; 0.001\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 139px;\"\u003e\n \u003cp\u003eCardiovagal CASS \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83px;\"\u003e\n \u003cp\u003e0.0 [0.0,1.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e1.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.0, 2.0]\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e1.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.0, 1.0]\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.0, 0.75]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.0, 1.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.161\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e0.198\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eBRS-V, ms/mmHg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83px;\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003cp\u003e[0.0,17.7]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.9\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[1.4, 17.5] \u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e8.4\u003c/p\u003e\n \u003cp\u003e[3.5, 13.3]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e8.1\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[6.0, 14.3]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e8.0\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[4.1, 13.8]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.504\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e0.190\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 139px;\"\u003e\n \u003cp\u003eBRS-A, ms/mmHg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 83px;\"\u003e\n \u003cp\u003e13.1\u003c/p\u003e\n \u003cp\u003e[3.1,23.1]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e10.1\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[0.0, 21.5]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 80px;\"\u003e\n \u003cp\u003e13.3\u003c/p\u003e\n \u003cp\u003e[8.0, 19.7]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e13.6\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[8.7, 18.2]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e14.9\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e[11.3, 18.0]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e0.630\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e0.248\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\" valign=\"top\" style=\"width: 624px;\"\u003e\n \u003cp\u003eData presented is median [1q, 3q] or N (% of column)\u003c/p\u003e\n \u003cp\u003eAbbreviations: BRS-A: adrenergic baroreflex sensitivity; BRS-V: vagal baroreflex sensitivity\u0026nbsp;\u003c/p\u003e\n \u003cp\u003ea, comparison across four immunotypes\u003c/p\u003e\n \u003cp\u003eb, binary comparison between immunotype 1 and immunotypes 2-4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-autonomic-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"autr","sideBox":"Learn more about [Clinical Autonomic Research](http://link.springer.com/journal/10286)","snPcode":"10286","submissionUrl":"https://www.editorialmanager.com/autr/default2.aspx","title":"Clinical Autonomic Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"autonomic nervous system, dysautonomia, baroreflex, neuropathy, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-5504909/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5504909/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003ePre-clinical studies have demonstrated direct influences of the autonomic nervous system (ANS) on the immune system. However, it remains unknown if connections between the peripheral ANS and immune system exist in humans and contribute to the development of chronic inflammatory disease. This study had three aims: 1.) To examine the relationship between IL-6 and the parasympathetic/vagal component of baroreflex sensitivity (BRS-V) in people with HIV; 2.) To determine if the subtype and severity of HIV-autonomic neuropathy (AN) would predict distinct immunotypes; 3.) To compare the burden of non-AIDS-related co-morbidities between immunotypes.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e79 adult people with well-controlled HIV underwent a standard battery of autonomic function tests summarized as the Composite Autonomic Severity Score and vagal and adrenergic baroreflex sensitivity (BRS-V and BRS-A). Levels of immune biomarkers were measured in all participants using the Target 96 Inflammation Panel on the Olink proteomics platform and immunotypes were identified using unbiased, non-negative matrix factorization. Mass cytometry (CyTOF) was completed on a subset of participants with and without autonomic neuropathy (N\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFirst, we found reduced BRS-V predicted higher levels of IL-6 (p\u0026thinsp;=\u0026thinsp;0.002). Second, a pro-inflammatory immunotype defined by elevations in type 1 cytokines (IL-6, IL-17) and increased numbers of CD8\u0026thinsp;+\u0026thinsp;T-cells was associated with autonomic neuropathy characterized by deficits in sympathetic nervous system activity (aOR\u0026thinsp;=\u0026thinsp;4.7, p\u0026thinsp;=\u0026thinsp;0.017). This pro-inflammatory immunotype was older with a greater burden of co-morbidities\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eDeficits in the parasympathetic/cardiovagal and the sympathetic nervous system are associated with inflammation and disease burden in people living with HIV. Future longitudinal research is needed to examine causality.\u003c/p\u003e","manuscriptTitle":"The Autonomic Nervous System (ANS)-Immune Network in People Living With HIV","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-25 01:22:39","doi":"10.21203/rs.3.rs-5504909/v1","editorialEvents":[{"type":"communityComments","content":1},{"type":"reviewerAgreed","content":"","date":"2024-11-25T15:58:07+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-25T14:05:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-23T07:09:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical Autonomic Research","date":"2024-11-22T08:27:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-autonomic-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"autr","sideBox":"Learn more about [Clinical Autonomic Research](http://link.springer.com/journal/10286)","snPcode":"10286","submissionUrl":"https://www.editorialmanager.com/autr/default2.aspx","title":"Clinical Autonomic Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"22905849-72a4-48ce-b7bd-aedaf80c73f6","owner":[],"postedDate":"December 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-12T16:00:32+00:00","versionOfRecord":{"articleIdentity":"rs-5504909","link":"https://doi.org/10.1007/s10286-025-01129-5","journal":{"identity":"clinical-autonomic-research","isVorOnly":false,"title":"Clinical Autonomic Research"},"publishedOn":"2025-05-10 15:56:50","publishedOnDateReadable":"May 10th, 2025"},"versionCreatedAt":"2024-12-25 01:22:39","video":"","vorDoi":"10.1007/s10286-025-01129-5","vorDoiUrl":"https://doi.org/10.1007/s10286-025-01129-5","workflowStages":[]},"version":"v1","identity":"rs-5504909","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5504909","identity":"rs-5504909","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}