BACKGROUND: We previously showed in a rat model of heart failure with preserved ejection fraction (HFpEF) that transcutaneous vagus nerve stimulation (tVNS) reduced cardiac fibrosis and inflammation. However, macrophage-mediated mechanisms through which tVNS rescues cardiac function remain poorly understood. METHODS: We induced HFpEF in 8-week-old mice by a combination of a high-fat diet and l -NG-nitro arginine methyl ester for 5 weeks, followed by 4 weeks of tVNS or sham stimulation. At this time, we analyzed cardiac function by echocardiography and immune cell numbers by single-cell RNA sequencing and flow cytometry. RESULTS: Our data demonstrate that HFpEF mice exhibited diastolic dysfunction, left ventricular hypertrophy, and fibrosis, consistent with HFpEF, and that tVNS significantly improved HFpEF severity. Analysis of merged single-cell RNA sequencing data from control, HFpEF+sham, and HFpEF+tVNS mice showed that HFpEF was associated with the accumulation of Spp1 -expressing CCR2 (C-C chemokine receptor type 2) ⁺ cardiac resident macrophages (CRM). Furthermore, treatment with tVNS reduced the number of CCR2 ⁺ CRM and the expression of Spp1 while also inducing the expression of Igf1 in TLF ⁺ (Timd4 ⁺ [T-cell immunoglobulin and mucin domain containing 4 ⁺ ]/Lyve1 ⁺ [Lymphatic vessel endothelial hyaluronan receptor 1 ⁺ ]/Folr2 ⁺ [Folate receptor 2 ⁺ ]) and MHC2 ⁺ (Major histocompatibility complex 2 ⁺ ) CRM. Global deletion of Spp1 or blockade of CCR2 ⁺ CRM recruitment improved HFpEF, whereas TLF ⁺ /MHC2 ⁺ specific deletion of Igf1 reversed the protective effect of tVNS on HFpEF. The benefits of tVNS were also abolished in the setting of disrupted acetylcholine/α7nAChR (α7 nicotinic acetylcholine receptor) signaling, either via pharmacological inhibition of α7nAChR or choline acetyltransferase deletion in CD4 ⁺ (cluster of differentiation) T cells. CONCLUSIONS: Collectively, our data indicate that tVNS improves HFpEF by reducing Spp1 expressing CCR2 ⁺ CRM and inducing expression of proreparative Igf1 in TLF ⁺ /MHC2 ⁺ CRM. These effects are mediated through cholinergic signaling, highlighting a neuroimmune pathway in HFpEF.

Tumor-infiltrating nerves play critical roles in promoting tumor growth and progression; however, the mechanisms that drive tumor innervation remain unclear. Upon transformation, tumors recruit surrounding peripheral nerves into the tumor microenvironment (TME) to obtain their own innervation, a process called axonogenesis. While in vitro studies suggest tumor cell-derived neurotrophins, such as brain-derived neurotrophic factor (BDNF), drive axonogenesis, this has yet to be demonstrated in vivo. During wound healing, macrophages are the primary source of neurotrophins. Given the critical role of macrophages in breast cancer growth, we investigated whether these immune cells drive tumor axonogenesis in breast tumors in vivo. Syngeneic Py230 mouse triple-negative breast cancer (TNBC) cells were transplanted into intact mice and mice lacking immune-derived BDNF. Bone marrow-derived macrophages from either wild-type or immune-BDNF-deficient mice were transplanted into tumor-bearing recipients to determine if macrophage-derived BDNF was sufficient to restore tumor growth and innervation. We found that transplanted TNBC cannot grow in the absence of immune-derived BDNF, and that depletion of macrophages from the TME compromises tumor innervation. Remarkably, the introduction of wild-type macrophages restores tumor growth and innervation in mice lacking immune-derived BDNF, demonstrating that macrophages are both necessary and sufficient for tumor axonogenesis in vivo. In the absence of sensory tumor innervation, tumor growth was significantly reduced. Moreover, targeting BDNF signaling diminished TNBC growth and innervation. Our findings identify macrophages as the critical source of BDNF driving axonogenesis in breast cancer and suggest that selectively targeting BDNF signaling could provide a novel therapeutic strategy for treating TNBC through compromising tumor innervation.

Influenza A virus (IAV) causes seasonal epidemics annually and pandemics every few decades. Most antiviral treatments used for IAV are only effective if administered during the first 48 h of infection and antiviral resistance is possible. Therapies that can be initiated later during IAV infection and that are less likely to elicit resistance will significantly improve treatment options. Pyruvate, a key metabolite, and an end product of glycolysis, has been studied for many uses, including its anti-inflammatory capabilities. Sodium pyruvate was recently shown by us to decrease inflammasome activation during IAV infection. Here, we investigated sodium pyruvate’s effects on IAV in vivo. We found that nebulizing mice with sodium pyruvate decreased morbidity and weight loss during infection. Additionally, treated mice consumed more chow during infection, indicating improved symptoms. There were notable improvements in pro-inflammatory cytokine production (IL-1β) and lower virus titers on day 7 post-infection in mice treated with sodium pyruvate compared to control animals. As pyruvate acts on the host immune response and metabolic pathways and not directly on the virus, our data demonstrate that sodium pyruvate is a promising treatment option that is safe, effective, and unlikely to elicit antiviral resistance.

Adrenergic nerves innervate all secondary lymphoid organs, although the role of these nerves in adaptive immune responses is unclear. Systemic depletion of these nerves compromises germinal center formation after infection or vaccination. We have found that these nerves locally are required for optimal B cell responses to the large antigen keyhole limpet hemocyanin (KLH), but are dispensable for the B cell response to the small antigen Ovalbumin. After vaccination or infection the reticulin fiber network in the lymph node undergoes remodeling to allow high molecular weight particles, such as KLH, access to the conduit system when they are ordinarily restricted. In contrast, low molecular weight molecules like Ovalbumin always have access to the conduit system. This remodeling of the reticulin fiber network to allow permeability to large molecules was dependent upon the local adrenergic nerves. These findings may help explain why patients with nerve injuries are more likely to have recurrent infections.