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Client Publication | Nature Communications | Xiangya Second Hospital team led by Dr. Ruping Dai and Dr. Hui Li reveals the hypothalamic paraventricular nucleus CRH neurons’ regulatory pathway in acute lung injury

Release time：2025-10-31 09:59:36

Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are devastating inflammatory lung diseases characterized by high morbidity and mortality, with limited effective treatments. The inflammatory mechanisms underlying ALI are complex, and neuroimmune interactions play a key role in maintaining pulmonary homeostasis. However, the specific brain regions involved in regulating lung inflammation remain unclear.

https://www.nature.com/articles/s41467-025-63953-7

On October 6, 2025, the research team led by Dr. Ruping Dai and Dr. Hui Li from Xiangya Second Hospital of Central South University published a paper in Nature Communications titled “Paraventricular nucleus CRH neurons regulate acute lung injury via sympathetic nerve–neutrophil axis.”

Using male mouse models combined with anatomical tracing, chemogenetic manipulation, and pharmacological intervention, the study identified a hypothalamic paraventricular nucleus CRHᴾⱽᴺ neuron–lung neural circuit that becomes active during ALI. Activation of these neurons protected mice from ALI, increased survival rates, and reduced neutrophil infiltration in lung tissue, while inhibition worsened the injury.

This protective effect was mediated by enhanced sympathetic nerve activity, in which locally released norepinephrine acted through the β2-adrenergic receptor–β-arrestin2 signaling pathway to modulate neutrophil function and inhibit the NF-κB pathway.

These findings provide crucial theoretical support for understanding how the central nervous system actively regulates ALI and offer new insights into potential therapeutic targets for ALI and ARDS.

Diagram: Mechanism by which activation of CRHᴾⱽᴺ neurons alleviates ALI

ALI Activates Hypothalamic Paraventricular Nucleus Corticotropin-Releasing Hormone (CRHᴾⱽᴺ) Neurons

In this study, an ALI and acute respiratory distress syndrome (ARDS) mouse model was established by intranasal instillation of lipopolysaccharide (LPS) to investigate the brain’s regulatory role in pulmonary injury (Fig. 1a).

Three hours after LPS exposure, c-Fos immunostaining revealed neuronal activation in six brain regions, including the paraventricular nucleus (PVN) of the hypothalamus. Nearly 80% of CRH⁺ neurons in the PVN were co-localized with c-Fos⁺ signals (Fig. 1b–e).

To further confirm that LPS activates PVN CRHᴾⱽᴺ neurons, AAV-EF1α-DIO-GCaMP6m was injected into the PVN of CRH-IRES-Cre mice, and calcium signals were monitored through fiber photometry. The results showed that LPS exposure significantly enhanced the calcium activity of CRHᴾⱽᴺ neurons (Fig. 1f–j).

To determine whether PVN CRHᴾⱽᴺ neurons project efferent signals to the lung, AAV2/9-Ef1α-DIO-mCherry was injected into the PVN of CRH-IRES-Cre mice to visualize CRHᴾⱽᴺ neurons. Three weeks later, a pseudorabies virus (PRV) expressing GFP was injected into the lungs for retrograde tracing. Six days post-injection, robust GFP signals were detected in numerous CRH neurons within the PVN, confirming a neuroanatomical connection between PVN CRHᴾⱽᴺ neurons and the lung (Fig. 1k–m).

Figure 1. Activation of CRHᴾⱽᴺ neurons by ALI.

Activation of CRHᴾⱽᴺ Neurons Alleviates ALI and Improves Survival, While Inhibition Exacerbates ALI and Increases Mortality

To investigate the regulatory role of CRHᴾⱽᴺ neurons in ALI, the researchers employed chemogenetic approaches. Excitatory AAV (hM3Dq) or control virus (Ctrl) was injected into the PVN of CRH-IRES-Cre mice (Fig. 2a). Continuous activation of CRHᴾⱽᴺ neurons for five days significantly improved survival rates (Fig. 2b), while two days of activation attenuated lung injury (Fig. 2c–d), reduced lung wet/dry (W/D) ratio and bronchoalveolar lavage fluid (BALF) protein concentration, thereby suppressing increased pulmonary permeability and edema (Fig. 2e–f). Moreover, proinflammatory cytokines such as IL-1β, IL-6, and TNF were markedly decreased in lung tissue (Fig. 2g).

Conversely, injection of an inhibitory AAV (hM4Di) into the PVN (Fig. 2h), followed by intraperitoneal administration of CNO to suppress neuronal activity in an ALI model induced by 10 mg/kg LPS (a dose chosen to avoid lethality that might mask the effect), resulted in significantly increased mortality (Fig. 2i). These mice also showed worsened lung inflammation and higher histopathological injury scores, with increased vascular permeability, aggravated edema (Fig. 2j–m), and elevated expression of IL-6, IL-1β, and TNF in lung tissues (Fig. 2n).

Collectively, these findings demonstrate that activation of CRHᴾⱽᴺ neurons mitigates ALI and enhances survival, whereas inhibition aggravates lung injury and increases mortality.

Figure 2. Regulation of ALI and survival rate by CRHᴾⱽᴺ neurons.

CRHᴾⱽᴺ Neurons Regulate Neutrophil Infiltration and Function During ALI

Given the pivotal role of neutrophils in inflammation-related ALI, the study analyzed changes in leukocytes (including neutrophils) within the BALF and lung tissue of LPS-treated mice following activation or inhibition of CRHᴾⱽᴺ neurons.

Upon activation of these neurons, flow cytometry revealed that CD45⁺ leukocyte and Ly6G⁺ neutrophil infiltration in the BALF and lungs were significantly reduced at both 8 and 48 hours after LPS exposure (Fig. 3a–c). Immunohistochemistry showed that the accumulation of Ly6G⁺ neutrophils and myeloperoxidase (MPO)-positive cells—which contribute to oxidative tissue injury—was suppressed in lung tissue (Fig. 3d–e). RNA sequencing of lung samples demonstrated downregulation of neutrophil-related genes and cytokine signaling pathways (Fig. 3f–g). In contrast, inhibition of CRHᴾⱽᴺ neurons increased neutrophil infiltration and inflammatory responses (Fig. 3h–l).

Together, these findings indicate that CRHᴾⱽᴺ neurons modulate the recruitment of neutrophils to the lungs during ALI.

Figure 3. CRHᴾⱽᴺ neurons influence neutrophil infiltration and response.

To further explore how CRHᴾⱽᴺ neurons regulate neutrophil function, RNA sequencing was performed on neutrophils isolated from the lungs of LPS-treated mice after chemogenetic activation of CRHᴾⱽᴺ neurons (Fig. 4a). Compared with control mice, 1,109 genes were downregulated and 1,028 genes were upregulated (Fig. 4b). KEGG pathway analysis indicated enrichment in pathways such as chemokine signaling and the NF-κB signaling pathway (Fig. 4c).

Functionally, activation of CRHᴾⱽᴺ neurons significantly suppressed the phagocytic ability of neutrophils in BALF (as shown by reduced uptake of FITC-labeled latex beads; Fig. 4d) and decreased reactive oxygen species (ROS) production (reduced DCFH-DA fluorescence intensity; Fig. 4e). Moreover, activation markedly inhibited the formation of neutrophil extracellular traps (NETs) in the lungs during ALI, as confirmed by co-localization of CitH3 and MPO staining (Fig. 4f–g). Collectively, these results demonstrate that activation of CRHᴾⱽᴺ neurons suppresses the pathogenic functions of neutrophils during ALI.

Figure 4. Activation of CRHᴾⱽᴺ neurons restricts neutrophil effector functions in ALI.

Pulmonary Sympathetic Nerves Mediate the Protective Effects of CRHᴾⱽᴺ Neurons on ALI via β₂-Adrenergic Receptors

The sympathetic nervous system (SNS) and the hypothalamic–pituitary–adrenal (HPA) axis are central pathways through which CRHᴾⱽᴺ neurons mediate neuroimmune interactions. Although activation of CRHᴾⱽᴺ neurons had different effects on plasma corticosterone levels (elevated under basal conditions but reduced following LPS treatment, reflecting HPA axis modulation), it consistently increased norepinephrine (NE) levels in lung tissue (Fig. 5a–b). Furthermore, CRHᴾⱽᴺ neuron activation upregulated tyrosine hydroxylase (TH) — the rate-limiting enzyme in catecholamine synthesis (Fig. 5c) — indicating enhanced pulmonary SNS activity.

Subsequent experiments demonstrated that nasal instillation of 6-hydroxydopamine (6-OHDA), which ablated pulmonary sympathetic nerves (Fig. 5d), or administration of the β₂-adrenergic receptor (β₂-AR) antagonist ICI118551 to block β₂-AR signaling in the lung (Fig. 5m), abolished the protective effects of CRHᴾⱽᴺ neuron activation — including improved survival, reduced leukocyte infiltration, and attenuated lung injury (Fig. 5g–j, 5k–l, 5n–r).

Collectively, these findings demonstrate that the pulmonary sympathetic nervous system and β₂-adrenergic receptors are essential mediators of the CRHᴾⱽᴺ neuron–induced protection against ALI.

Figure 5. CRHᴾⱽᴺ neurons regulate ALI via local sympathetic nerve activation.

Norepinephrine Regulates Neutrophil Function via the β₂-AR–β-arrestin2 Signaling Pathway to Inhibit the NF-κB Pathway

To clarify the effect of the pulmonary SNS on neutrophil function, the study examined the influence of NE on bone marrow–derived neutrophils in vitro. The results showed that NE reduced LPS-induced phagocytosis of neutrophils (Fig. 6b), decreased reactive oxygen species (ROS) production (Fig. 6c), and suppressed TNF expression (Fig. 6f). Similarly, the β₂-adrenergic receptor (β₂-AR) agonist salbutamol produced comparable inhibitory effects (Fig. 6d–e), suggesting that NE suppresses neutrophil responses to LPS through β₂-AR signaling.

RNA sequencing of lung neutrophils from LPS-treated control mice and hM3Dq-expressing mice (with activated CRHᴾⱽᴺ neurons) identified the NF-κB signaling pathway as a key regulatory pathway modulated by CRHᴾⱽᴺ neuron activation (Fig. 6g). Mechanistically, NE inhibited the phosphorylation of p65 and IκBα in LPS-stimulated neutrophils (Fig. 6h), reduced nuclear translocation of phosphorylated p65 (Fig. 6i), and upregulated β-arrestin2, a negative regulator of NF-κB signaling downstream of β₂-AR activation (Fig. 6j).

Taken together, these findings indicate that NE primarily modulates neutrophil function through a β₂-AR/β-arrestin2–dependent inhibition of the NF-κB pathway, representing a potential mechanism by which CRHᴾⱽᴺ neuron activation exerts its protective effects against ALI.

Figure 6. NE regulates neutrophils via the β₂-AR–β-arrestin2 signaling pathway to inhibit the NF-κB pathway.

Conclusion

This study is the first to reveal the existence of a “CRHᴾⱽᴺ neuron–pulmonary sympathetic nerve–neutrophil” axis. Activation of CRHᴾⱽᴺ neurons enhances pulmonary sympathetic nerve activity, leading to the release of NE. NE binds to β₂-adrenergic receptors (β₂-AR) on neutrophils and activates β-arrestin2, thereby inhibiting the NF-κB signaling pathway. This cascade ultimately reduces neutrophil infiltration and pathogenic activity, alleviating ALI.

Future studies should further investigate the roles of other brain regions and different neuronal subpopulations within the PVN in the regulation of pulmonary inflammation, to build a more comprehensive understanding of the brain–lung regulatory network. Long-term studies are also needed to monitor the dynamic activity of CRHᴾⱽᴺ neurons at various stages of ALI and to assess the safety and therapeutic efficacy of CRH neuron–based interventions, potentially in combination with noninvasive neuromodulation technologies.

All viral tools used in this study are available
from Brain Case Biotech
（bd@ebraincase.com)

Product Category	Product No.	Product Name
Fluorescent Protein	BC-0016	rAAV-EF1α-DIO-mCherry
Chemogenetic Inhibition	BC-0155	rAAV-EF1α-DIO-hM4D(Gi)-mCherry
	BC-0146	rAAV-EF1α-DIO-hM3D(Gq)-mCherry
Calcium Imaging	BC-0087	rAAV-EF1α-DIO-GCaMP6m
PRV	BC-PRV-531	PRV-CAG-EGFP

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Client Publication | Nature Communications | Xiangya Second Hospital team led by Dr. Ruping Dai and Dr. Hui Li reveals the hypothalamic paraventricular nucleus CRH neurons’ regulatory pathway in acute lung injury

ALI Activates Hypothalamic Paraventricular Nucleus Corticotropin-Releasing Hormone (CRHᴾⱽᴺ) Neurons

Activation of CRHᴾⱽᴺ Neurons Alleviates ALI and Improves Survival, While Inhibition Exacerbates ALI and Increases Mortality

CRHᴾⱽᴺ Neurons Regulate Neutrophil Infiltration and Function During ALI

Pulmonary Sympathetic Nerves Mediate the Protective Effects of CRHᴾⱽᴺ Neurons on ALI via β₂-Adrenergic Receptors

Norepinephrine Regulates Neutrophil Function via the β₂-AR–β-arrestin2 Signaling Pathway to Inhibit the NF-κB Pathway

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Client Publication | Nature Communications | Xiangya Second Hospital team led by Dr. Ruping Dai and Dr. Hui Li reveals the hypothalamic paraventricular nucleus CRH neurons’ regulatory pathway in acute lung injury

ALI Activates Hypothalamic Paraventricular Nucleus Corticotropin-Releasing Hormone (CRHᴾⱽᴺ) Neurons

Activation of CRHᴾⱽᴺ Neurons Alleviates ALI and Improves Survival, While Inhibition Exacerbates ALI and Increases Mortality

CRHᴾⱽᴺ Neurons Regulate Neutrophil Infiltration and Function During ALI

Pulmonary Sympathetic Nerves Mediate the Protective Effects of CRHᴾⱽᴺ Neurons on ALI via β₂-Adrenergic Receptors

Norepinephrine Regulates Neutrophil Function via the β₂-AR–β-arrestin2 Signaling Pathway to Inhibit the NF-κB Pathway

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