Background: Heart failure with preserved ejection fraction (HFpEF) is a rapidly growing clinical problem with limited therapeutic options. Although patients present with diverse extracardiac comorbidities such as obesity, these conditions share a common feature: they induce systemic low-grade inflammation and metabolic stress. Given that immune cells circulate within this inflammatory milieu, they offer a direct window into disease-related immune mechanisms, enabling characterization of HFpEF-associated immune activation and identification of targetable pathways. Nitro-oleic acid (NO₂-OA) is an endogenously formed electrophilic fatty acid that modulates cellular signaling through reversible protein modification. In humans, NO₂-OA has demonstrated safety and reduced circulating cytokine levels in obese individuals, which has led to ongoing clinical development. In this study, we characterized the effects of HFpEF, relative to HFrEF (reduced EF), on circulating immune cells and assessed whether their inflammatory and metabolic activation state can be modulated by NO₂-OA.
Methods: 158,802 peripheral blood mononuclear cells (PBMCs) from patients with HFpEF (n=6), HFrEF (n=8), and controls (n=7) +/- obesity were analyzed by single-cell RNA sequencing. Key findings were validated in an independent whole-blood bulk RNA seq cohort (HFpEF n=18, HFrEF n=16, controls n=8). Cross-species conservation was assessed by profiling 57,097 PBMCs from a diet+L-NAME HFpEF mouse model +/- NO₂-OA treatment. NO₂-OA effect on human cells was tested by analyzing cytokine-stimulated PBMCs from healthy donors +/- prior NO₂-OA treatment.
Results: HFpEF PBMCs exhibited a distinct immune activation profile compared to HFrEF PBMCs. Both HF-groups showed NK and T cell activation, yet HFpEF preserved balanced monocyte/T cell proportions, whereas HFrEF shifted toward increased monocyte and reduced T cell fractions. HFpEF PBMCs demonstrated obesity-dependent inflammatory differences: lean HFpEF PBMCs showed higher CCL2, obese HFpEF PBMCs increased TNF expression. HFrEF PBMCs, however, displayed a different inflammatory transcriptomic profile, including complement pathway activation, indicating a distinct inflammatory architecture. Beyond inflammation, HFpEF PBMCs showed increased expression of mitochondrial-associated genes (e.g., ATP5ME, UQCRQ), suggesting metabolic activation not observed in HFrEF PBMCs. These HFpEF-specific signatures were conserved in the murine HFpEF model, allowing translational testing of pathway modulation. PBMCs from HFpEF mice showed elevated expression of inflammatory and metabolic stress genes. Treatment with NO₂-OA reduced these responses and improved diastolic function. To assess direct effects of NO₂-OA on human immune cells, PBMCs from healthy donors were stimulated with pro-inflammatory cytokines. Pre-treatment with NO₂-OA attenuated induction of inflammatory and metabolic stress genes (IL1B, CASP1, OLR1, INSIG1) and reduced NF-κB phosphorylation and IL-1β maturation, demonstrating that NO₂-OA directly modulates immune activation in human PBMCs.
Conclusion: HFpEF PBMCs exhibit a unique immunometabolic activation pattern that differs from HFrEF. NO₂-OA attenuates inflammatory and metabolic responses in human and murine PBMCs, supporting immunometabolic pathway modulation as a potential target for future therapeutic approaches in HFpEF. Nitro-oleic acid may be a candidate for further testing.
