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Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF) -a deadly disease with restricted and limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECII. Deficiency of CYB5R3 in AECII leads to sustained activation of the profibrotic factor TGF-β1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and sGC-cGMP-protein kinase G axis that modulates activation of TGF-β1 signaling pathway. We demonstrate that sGC agonists are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECII. Taken together, these results establish that CYB5R3 in AECII is required to maintain resilience against lung injury and fibrosis, and that therapeutic manipulation of sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.

Maternal COVID-19 vaccination could protect infants who are ineligible for vaccine through antibody transfer during pregnancy and lactation. We measured the quantity and durability of SARS-CoV-2 antibodies in human milk and infant blood before and after maternal booster vaccination. Prospective cohort of lactating women immunized with primary and booster COVID-19 vaccines during pregnancy or lactation and their infants. Milk and blood samples from October 2021 to April 2022 were included. Anti-nucleoprotein (NP) and anti-receptor binding domain (RBD) IgG and IgA in maternal milk and maternal and infant blood were measured and compared longitudinally after maternal booster vaccine. Forty-five lactating women and their infants provided samples. 58% of women were anti-NP negative and 42% were positive on their first blood sample prior to booster vaccine. Anti-RBD IgG and IgA in milk remained significantly increased through 120–170 days after booster vaccine and did not differ by maternal NP status. Anti-RBD IgG and IgA did not increase in infant blood after maternal booster. Of infants born to women vaccinated in pregnancy, 74% still had positive serum anti-RBD IgG measured on average 5 months after delivery. Infant to maternal IgG ratio was highest for infants exposed to maternal primary vaccine during the second trimester compared to third trimester (0.85 versus 0.29; p<0.001). Maternal COVID-19 primary and booster vaccine resulted in robust and long-lasting transplacental and milk antibodies. These antibodies may provide important protection against SARS-CoV-2 during the first six months of life.

Backgound Alterations in the redox homeostasis play a role in the pathogenesis of age‐related diseases such idiopathic pulmonary fibrosis (IPF). The most accepted theory in the pathogenesis of IPF is the injury and activation of alveolar epithelial type II cells (AECII) triggering excessive and chronic wound‐healing responses, mediated by growth factors such as TGF‐β. A mitochondrial membrane‐bound NADH‐dependent redox enzyme that contributes to stress protection and associates with healthspan and aging is cytochrome b5 reductase isoform 3 (Cyb5R3). Cyb5R3 is a flavoprotein recognized for its ability to reduce heme iron, a major target of reactive oxygen species. However, the link between Cyb5R3 expression levels in AECII with the modulation of the TGF‐β signaling pathways and the age‐related vulnerability to injury and fibrosis in the lung is unknown. Methods Lungs from young, old and IPF donors were lysated to test Cyb5R3 levels. Cyb5R3 flox mice (Cyb5R3 fl/fl or WT) and mice carrying a doxycycline‐inducible Cre‐recombinase under the control of a regulatory sequence of the SP‐C gene were bred. To promote recombination and generate a homozygous conditional type II lung epithelial cells (AECII) CYB5R3 knockout mice (AECII Cyb5R3 KO), mice were fed doxycycline chow (625 mg/kg, Envigo) for 2 weeks before injury or AECII isolation. Mice were infected intranasally with gammaherpesvirus 68 (MHV68) to evaluate inflammatory and fibrotic responses in the lung. Mouse alveolar epithelial cell line MLE12 was used to knockdown Cyb5R3 using adenovirus carrying scrambleshRNA or Cyb5R3shRNA constructs. Results Aged donor and IPF total lung lysates show lower levels of Cyb5R3. In addition, isolated murine primary AECII showed that Cyb5R3 is significantly diminished in aging AECIIs (24 mo vs. 4 mo) associated with cellular oxidative stress and a senescence phenotype. Conditional deletion of Cyb5R3 in AECII show increased susceptibility to lung fibrosis. AECII Cyb5R3 KO mice showed higher collagen deposition with significant higher transcript levels of TIMP1 (tissue inhibitor of the MMPs) and osteopontin than Cyb5R3 WT mice. Since TGF‐β is a key modulator of the net balance between MMPs and TIMPs, we analyzed the role of Cyb5R3 in TGF‐β signaling. In mouse epithelial cells, deficiency of Cyb5R3 enhances TGF‐β signaling with upregulation of profibrotic and senescence genes by modulation of cGMP‐PKG and cAMPPKA pathways. Conclusions Altogether, these data suggest a possible role for Cyb5R3 in the fragility of AECIIs and the age related susceptibility to lung fibrosis. New therapies designed to modulate Cyb5R3 expression and activity might lessen severity of pulmonary fibrosis, especially in the context of the aging lung. Support or Funding Information This work was funded by the Aging Institute & Vascular Medicine Institute at the University of Pittsburgh