OBJECTIVE:Microbial small RNAs can regulate human genes. Higher plasma concentrations of microbial transfer RNA (tRNA)-derived RNA-1 (tDR-1) were previously associated with lower rheumatoid arthritis (RA) disease activity. This study examined whether tDR-1 concentrations differ in anticyclic citrullinated peptide-3 positive (CCP3+) at-risk individuals (ARI), based on who later develops clinical RA, or discriminate later conversion status beyond established risk factors and if tDR-1 has in vitro effects. METHODS:Plasma tDR-1 concentrations were measured in CCP3+ ARI. Group differences in log-transformed tDR-1 were assessed by lognormal Welch's t-test and logistic regression. Area under the receiver operating characteristic curve (AUROC) was used to evaluate discriminatory ability. Human THP-1 monocyte-derived macrophages were treated with tDR-1 versus scramble control, and gene expression was assessed by NanoString Immunology panel. RESULTS:Among 60 CCP3+ ARI, 25 later developed clinical RA ("converters") over a mean of 2.2 years, whereas 35 did not ("nonconverters") over a mean of 5.3 years. Baseline plasma tDR-1 concentrations were significantly higher (5.4-fold) in nonconverters versus converters, even after adjustment for additional RA risk factors (shared epitope, smoking, rheumatoid factor) (P = 5.1 × 10-4). The AUROC improved from 0.722 for these risk factors alone to 0.902 with the addition of tDR-1 (P = 0.003). In vitro, tDR-1 significantly down-regulated many type 1 interferon (IFN) response genes in THP-1 cells. CONCLUSION:Higher plasma tDR-1 concentrations were associated with nonconversion to clinical RA in CCP3+ ARI. tDR-1's reducing type 1 IFN response gene expression suggests a potential mechanism by which microbes and tDR-1 could affect RA development.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are prevalent joint diseases, yet early diagnosis remains challenging with existing methods. Circulating microRNAs are promising biomarkers for detection and differentiation of arthritis subtypes. This study aimed to profile plasma microRNAs from early OA (N = 22), early RA (N = 12), and non-OA/RA (N = 50) individuals using microRNA-sequencing. Principal component analysis revealed distinct clustering of early OA from both early RA and non-OA/RA, but not for early RA and non-OA/RA. A total of 170 differentially expressed microRNAs were identified in early OA versus the other groups, with no significant differences found between early RA and non-OA/RA. Stepwise filtering followed by RT-qPCR validation in independent samples identified six microRNAs: miR-16-5p and miR-29c-3p were upregulated in early OA compared to both early RA and non-OA/RA, while miR-744-5p, miR-382-5p, miR-3074-5p, and miR-11400 were upregulated in early RA compared to the other two groups. Additionally, three novel microRNAs were identified using bioinformatic tools—one enriched in early OA and two in early RA. Target prediction and pathway analyses revealed that early OA microRNAs were linked to extracellular matrix degradation pathways, and early RA microRNAs were linked to immune signaling. These findings highlight six known and three novel circulating microRNAs with potential as biomarkers to distinguish early OA from early RA.

Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular disease (CVD), even in normotensive people. Systemic lupus erythematosus (SLE) is also a risk factor for CVD and hypertension. SLE and CVD are on the rise, and it is not known if this is attributable to the increased dietary salt in the modern diet and SSBP. Isolevuglandins (IsoLGs) adduct self-proteins forming neoantigens in antigen-presenting cells (APC), activate T cells, and produce cytokines that promote sodium retention, kidney damage, SLE, and SSBP. We hypothesized the adoptive transfer of peripheral blood mononuclear cells (PBMCs) from patients with SLE into immunodeficient mice (NSG/MHCI/MHCII-/-) would increase inflammation and SSBP after high salt feeding. In vitro studies were performed using PBMCs from patients with SLE and treated with and without high salt. Myeloid cell activation and cytokine expression were measured with flow cytometry. We humanized mice with PBMCs from patients with SLE (n=7) and controls without autoimmune disease (n=7) in animal studies. The mice were fed a 4% high-salt diet for two weeks. Blood pressure was measured with radiotelemetry, immune phenotyping was performed with flow cytometry, and vascular reactivity was assessed in mesenteric arteries using wire myography. A high salt diet significantly increased systolic blood pressure in lupus mice compared to the controls (133.0 vs.116.4 mmHg, p=0.001). APC infiltration (macrophages, monocytes, dendritic cells), proinflammatory markers, and oxidative stress (Isolevuglandins, Nox2) were significantly higher in the kidneys of lupus mice. In lupus mice, classical monocyte and proinflammatory markers were significantly elevated in the spleen lymphocytes, and proinflammatory markers were increased in the kidney, spleen, and aorta in lupus mice. These mice also exhibited mesenteric artery vascular dysfunction (p<0.05). Lupus mice tended to excrete more urinary albumin than the controls (25.89 vs. 17.24 µg/ml p=0.142), suggesting kidney injury. High salt treatment in vitro increased intermediate and nonclassical monocytes with increased CD86, IsoLG, and TNF-alpha expression compared to normal salt-treated cells. Mitochondria 3D analysis showed morphological changes such as increased mitochondrial fragmentation in high cells treated monocytes. In conclusion, our findings suggest that immune cells from patients with SLE, in response to high salt, increase SSBP and the proinflammatory milieu in the kidney, aorta, and spleen; impair vascular function; change mitochondrial morphology, and worsen albuminuria in immunodeficient mice. This study was supported by the National Institutes of Health grants R01 HL144941 (AK), and American Heart Association 23CDA1053072 (MS). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

OBJECTIVE:The prevalence of hypertension, a major cardiovascular risk factor, is increased in patients with rheumatoid arthritis (RA) and may be driven by immune activation. The purpose of this study was to determine if ambulatory 24-hour blood pressure (BP) is elevated in RA vs control participants and whether it is associated with immune activation. METHODS:We conducted a cross-sectional study of 46 patients with RA and 23 control participants. Participants wore an ambulatory BP monitor that obtained diurnal BP every 15-30 minutes and nocturnal BP every 30 minutes. Inflammatory mediators in plasma were measured using an inflammation proteomics panel. Differences in BP measurements were assessed by Mann-Whitney U test, and association with inflammatory mediators was assessed by Spearman correlation. RESULTS:Patients with RA and control participants had similar office BP, but median ambulatory systolic BP (SBP) measurements (24-hour [RA 121 mmHg vs control 116 mmHg; P = 0.01], diurnal [RA 128 mmHg vs control 120 mmHg; P = 0.003], and nocturnal [RA 112 mmHg vs control 103 mmHg; P = 0.002]) were higher in patients with RA. Patients with RA also had higher nocturnal diastolic BP (DBP; RA 63 mmHg vs control 57 mmHg; P = 0.02), but other DBP measurements were similar. Nocturnal BP dipping was less in patients with RA (12%) compared to control participants (16%; P = 0.02). In patients with RA, higher 24-hour and nocturnal SBPs and less nocturnal dipping were strongly correlated with a wide range of inflammatory mediators. CONCLUSION:Despite similar office measurements, 24-hour and nocturnal SBP measurements were higher in patients with RA than in control participants and were strongly associated with inflammation.

Objective High‐density lipoprotein (HDL) has well‐characterized anti‐atherogenic cholesterol efflux and antioxidant functions. Another function of HDL uncharacterized in rheumatoid arthritis (RA) is its ability to transport microRNAs (miRNAs) between cells and thus alter cellular function. The study's purpose was to determine if HDL‐miRNA cargo is altered and affects inflammation in RA. Methods HDL‐microRNAs were characterized in 30 RA and 30 control participants by next generation sequencing and quantitative polymerase chain reaction. The most abundant differentially expressed miRNA was evaluated further. The function of miR‐1246 was assessed by miRNA mimics, antagomiRs, small interfering RNA knockdown, and luciferase assays. Monocyte‐derived macrophages were treated with miR‐1246‐loaded HDL and unmodified HDL from RA and control participants to measure delivery of miR‐1246 and its effect on interleukin‐6 (IL‐6). Results The most abundant miRNA on HDL was miR‐1246; it was significantly enriched two‐fold on HDL from RA versus control participants. HDL‐mediated miR‐1246 delivery to macrophages significantly increased IL6 expression 43‐fold. miR‐1246 delivery significantly decreased DUSP3 1.5‐fold and DUSP3 small interfering RNA knockdown increased macrophage IL6 expression. Luciferase assay indicated DUSP3 is a direct target of miR‐1246. Unmodified HDL from RA delivered 1.6‐fold more miR‐1246 versus control participant HDL. Unmodified HDL from both RA and control participants attenuated activated macrophage IL6 expression, but this effect was significantly blunted in RA so that IL6 expression was 3.4‐fold higher after RA versus control HDL treatment. Conclusion HDL‐miR‐1246 was increased in RA versus control participants and delivery of miR‐1246 to macrophages increased IL‐6 expression by targeting DUSP3 . The altered HDL‐miRNA cargo in RA blunted HDL's anti‐inflammatory effect. image

Objective: Rheumatoid arthritis (RA) is a systemic autoimmune disease with complex pathogenesis involving the innate and adaptive immune system. Clonal hematopoiesis of indeterminate potential (CHIP) is defined by clonal proliferation of one hematopoietic stem cell and is typically asymptomatic. Both are common among older adults. CHIP is associated with multiple autoimmune diseases, but has not been thoroughly evaluated for its relationship with RA. Methods: We examined three large biobanks where CHIP status of participants has been determined from whole genome sequencing data. We ascertained cases of RA, seropositive RA (SPRA), and seronegative RA (SNRA) using established methods and used survival analysis to test whether CHIP status was predictive of incident disease. We combined the results of the three biobank studies using random effects meta-analysis. For validation, we performed deep, targeted sequencing of CHIP-causing genes in an established clinical cohort of 132 RA cases, 56 controls, and 544 external controls. We compared the rates of CHIP between cases and controls using logistic linear regression. Results: In the UKBiobank and in meta-analysis, the presence of a large CHIP clone was associated with an increased risk for SPRA (HR = 2.57 with CI [1.46, 4.52] and p = 0.001) and RA (HR = 1.43 with CI [1.16, 1.75] and p = 7 x 10-4). Medium CHIP clones were associated with smaller increases in risk for SPRA and RA, and small CHIP clones carried no increased risk of any outcome. There were no associations detected between SNRA and CHIP of any size. In the clinical RA cohort, cases were more likely to have CHIP than controls after correcting for age, age2, and sex (OR: 2.08, HR [1.09, 3.83], p = 0.02). Conclusion: In a meta-analysis combing data from three large biobanks, large CHIP clones were associated with an increased risk for incident SPRA and, to a lesser extent, increased risk for RA. Validating this biobank-based finding, in a well-phenotyped clinical cohort, cases had higher rates of CHIP than age-matched controls. The mechanism by which CHIP drives the increased risk for SPRA is not known, but if it were discovered, could inform early intervention for patients with CHIP to prevent RA or personalized therapy for patients with RA based on CHIP status. ### Competing Interest Statement AGB: Advisory board & Equity holder: TenSixteen Bio. ### Funding Statement This work was supported by NIH DP5 OD029586, a Burroughs Wellcome Fund Career Award for Medical Scientists, an E.P. Evans Foundation grant, a RUNX1 Research Program grant, a Pew Charitable Trusts and Alexander and Margaret Stewart Trust Pew-Stewart Scholar for Cancer Research award, a Vanderbilt University Medical Center Brock Family Endowment grant, and a Young Ambassador Award (A.G.B.), NIH T32 GM007347 (Y.P.), NIH T32 AR059039 (R.W.C.), and Arthritis National Research Foundation grant 128808 (R.W.C.). We would also like to acknowledge DNANexus for providing cloud computing credits. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: IRB of Vanderbilt University Medical Center gave ethical approval for this work (IRB #000567) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors except those owned by the UKBiobank, NIH AllOfUs, and Vanderbilt BioVU consortia, which each have their own access protocols.

Patients with rheumatoid arthritis (RA) have increased hypertension. Tissue sodium may contribute to development and progression of hypertension through immune cell activation. This study aimed to determine if skin sodium content is: 1) higher in RA versus control participants, and 2) associated with blood pressure and disease activity. This cross-sectional study included 32 patients with RA and 33 control participants. Lower leg skin sodium content was measured using magnetic resonance imaging. Ambulatory 24-h blood pressure measurements were obtained, and disease activity was assessed by Disease Activity Score-28 for RA with CRP (DAS28-CRP). Skin sodium content was higher in RA versus control participants (14.22 [12.82, 18.04] vs 12.41 [10.67, 14.55] mmol/L), p = 0.005. Every 1 mmol/l increase in skin sodium was associated with a 1.05 mmHg (95% CI 0.29, 1.82 mmHg, p = 0.009) increase in average 24-h systolic blood pressure in patients with RA, but this relationship was not present in control participants. Skin sodium was not associated with DAS28-CRP or its components. Skin sodium is increased in RA versus control participants and is correlated with 24-h and diurnal systolic blood pressure in patients with RA but not in control participants. Skin sodium content may help explain increased hypertension in patients with RA.

Purpose (the aim of the study): In clinical settings, osteoarthritis (OA) and rheumatoid arthritis (RA) can present similarly and therefore can be difficult to distinguish, especially at early stages of single-joint disease when symptoms overlap. OA is most often assessed through radiography [e.g., Kellgren-Lawrence (KL) grading] by changes in bone (e.g., osteophytes) and joint space narrowing, features that are less obvious in early stages. Meanwhile, RA is diagnosed through symptom duration and serological factors which can overlap with other inflammatory conditions, especially in early stages.

ObjectiveHypertension is frequent in patients with systemic lupus erythematosus (SLE) and is a major contributor to increased cardiovascular risk. Isolevuglandins (IsoLGs) are downstream products of oxidative stress that drive hypertension and SLE disease activity in animal models. Antibodies to IsoLGs (anti-IsoLGs) are present in human SLE and associated with disease activity, but it is not known if concentrations are higher compared to control subjects or if they are associated with blood pressure (BP).MethodsWe measured serum anti-IsoLG IgG antibody concentrations by sandwich ELISA in 23 patients with SLE and 30 controls who had participated in a cross-sectional 24-hour ambulatory BP study. We examined the association between anti-IsoLG IgG antibodies and BP measurements in patients with SLE and controls by Spearman Rho (rs) and linear regression analysis.ResultsSerum anti-IsoLG IgG antibody concentrations were higher in patients with SLE than controls (P = 0.007) and inversely associated with BP in SLE but not controls. In patients with SLE antibody concentrations were inversely associated with office (rs = −0.418) and diurnal systolic BP (rs = −0.421); the relationship was stronger among patients not taking anti-hypertensives (office: rs = −0.740, diurnal systolic BP: rs = −0.802) and every 20% increase in antibody concentration was associated with 10 mmHg decrease in 24-hour systolic BP (P = 0.004).ConclusionSerum anti-IsoLG IgG antibody concentrations are higher in patients with SLE than controls and are inversely associated with 24-hour BP measurements. Since IsoLGs promote hypertension, it is possible that in SLE, IsoLG antibodies could help clear these hypertension-inducing antigens.

Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular disease (CVD), even in normotensive people. Systemic lupus erythematosus (SLE) is also a risk factor for CVD and hypertension. SLE and CVD are on the rise, and it is not known if this is attributable to the increased dietary salt in the modern diet and SSBP. Isolevuglandins (IsoLGs) adduct self-proteins forming neoantigens in antigen presenting cells (APC) to activate T cells and produce cytokines that promote sodium retention, kidney damage, salt sensitivity of blood pressure, and SLE. We hypothesized that high salt intake increases both blood pressure and the proinflammatory milieu in mice humanized with the immune cells of patients with SLE. We humanized female NSG/MHCI/MHCII−/− immunodeficient mice with peripheral blood mononuclear cells (PBMCs) isolated from patients with SLE (n=5) and controls without autoimmune disease (n=4). The blood pressure was measured with radiotelemetry, immune phenotyping was performed with flow cytometry, and vascular reactivity was assessed in mesenteric arteries using wire myography after two weeks of a 4% high salt diet. High salt treatment significantly increased systolic blood pressure in lupus mice compared to the controls (135.5 vs. 123.8 mmHg, p=0.014). APC infiltration (macrophages, monocytes, dendritic cells), their activation (CD86) and proinflammatory markers (IL-23 p19, TNF-alpha), and oxidative stress (Isolevuglandins, Nox2) were significantly higher in the kidneys of lupus mice. Moreover, the signaling molecule p-STAT3 was significantly higher in myeloid cells in the kidneys of lupus mice. The classical monocyte and proinflammatory markers were significantly elevated in the spleen lymphocytes, and proinflammatory markers were increased in the kidney, spleen, and aorta in lupus mice. These mice exhibited vascular dysfunction (p<0.05), suggesting reduced vascular compliance. We measured urine albumin and found that lupus mice tended to excrete more albumin than the controls (17.24 vs. 25.89 μg/ml p=0.142), suggesting greater kidney injury. In conclusion, our findings suggest that immune cells from patients with SLE, in response to high salt intake, increase blood pressure and the proinflammatory milieu in the kidney, aorta, and spleen; impair vascular compliance; and worsen albuminuria in immunodeficient mice. 1R01HL144941-01A1 1R03HL155041 23CDA1053072. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

T cells in systemic lupus erythematosus (SLE) exhibit multiple metabolic abnormalities. Excess iron can impair mitochondria and may contribute to SLE. To gain insights into this potential role of iron in SLE, we performed a CRISPR screen of iron handling genes on T cells. Transferrin receptor (CD71) was identified as differentially critical for T H 1 and inhibitory for induced regulatory T cells (iT regs ). Activated T cells induced CD71 and iron uptake, which was exaggerated in SLE-prone T cells. Cell surface CD71 was enhanced in SLE-prone T cells by increased endosomal recycling. Blocking CD71 reduced intracellular iron and mTORC1 signaling, which inhibited T H 1 and T H 17 cells yet enhanced iT regs . In vivo treatment reduced kidney pathology and increased CD4 T cell production of IL-10 in SLE-prone mice. Disease severity correlated with CD71 expression on T H 17 cells from patients with SLE, and blocking CD71 in vitro enhanced IL-10 secretion. T cell iron uptake via CD71 thus contributes to T cell dysfunction and can be targeted to limit SLE-associated pathology.

We describe a mechanism responsible for systemic lupus erythematosus (SLE). In humans with SLE and in 2 SLE murine models, there was marked enrichment of isolevuglandin-adducted proteins (isoLG adducts) in monocytes and dendritic cells. We found that antibodies formed against isoLG adducts in both SLE-prone mice and humans with SLE. In addition, isoLG ligation of the transcription factor PU.1 at a critical DNA binding site markedly reduced transcription of all C1q subunits. Treatment of SLE-prone mice with the specific isoLG scavenger 2-hydroxybenzylamine (2-HOBA) ameliorated parameters of autoimmunity, including plasma cell expansion, circulating IgG levels, and anti-dsDNA antibody titers. 2-HOBA also lowered blood pressure, attenuated renal injury, and reduced inflammatory gene expression uniquely in C1q-expressing dendritic cells. Thus, isoLG adducts play an essential role in the genesis and maintenance of systemic autoimmunity and hypertension in SLE.

Abstract Introduction Infectious diseases are common causes of morbidity and mortality worldwide. Susceptibility to infection is highly heritable; however, little has been done to identify the genetic determinants underlying common infectious diseases. One GWAS was performed using 23andMe information about self-reported infections; we set out to confirm previous loci and identify new ones using medically diagnosed infections. Methods We used the electronic health record (EHR)-based biobank at Vanderbilt and diagnosis codes to identify cases of 12 infectious diseases in white patients: urinary tract infection, pneumonia, chronic sinus infections, otitis media, candidiasis, streptococcal pharyngitis, herpes zoster, herpes labialis, hepatitis B, infectious mononucleosis, tuberculosis (TB) or a positive TB test, and hepatitis C. We selected controls from patients with no diagnosis code for the candidate disease and matched by year of birth, sex, and calendar year at first and last EHR visits. We conducted GWAS using SAIGE and transcriptome-wide analysis (TWAS) using S-PrediXcan. We also conducted phenome-wide association study to understand associations between identified genetic variants and clinical phenotypes. Results We replicated three 23andMe loci (p ≤ 0.05): herpes zoster and rs7047299-A (p = 2.6 × 10–3) and rs2808290-C (p = 9.6 × 10–3;); otitis media and rs114947103-C (p = 0.04). We also identified 2 novel regions (p ≤ 5 × 10–8): rs113235453-G for otitis media (p = 3.04 × 10–8), and rs10422015-T for candidiasis (p = 3.11 × 10–8). In TWAS, four gene-disease associations were significant: SLC30A9 for otitis media (p = 8.06 × 10–7); LRP3 and WDR88 for candidiasis (p = 3.91 × 10–7 and p = 1.95 × 10–6); and AAMDC for hepatitis B (p = 1.51 × 10–6). Conclusion We conducted GWAS and TWAS for 12 infectious diseases and identified novel genetic contributors to the susceptibility of infectious diseases.