Background Inflammatory bowel diseases (IBDs) are characterised by dysbiosis and a leaky gut. The NADPH oxidase dual oxidase 2 (DUOX2) is upregulated in patients with IBD, yet its role in driving the disease remains unclear. Objective We interrogated the functional consequences of epithelial DUOX2 activity for the host and microbiome. Design DUOX2 function was studied in mice with epithelial-specific DUOX2 overactivation (vTLR4), inactivation (vTLR4 DUOXA IEC-KO) and wild-type controls. We assessed the effect of dysbiosis on DUOX2 signalling and intestinal permeability (FITC-dextran, serum zonulin, bacterial translocation) with germ-free (GF) mice engrafted with IBD or healthy microbiota. RNA sequencing of colonic mucosa and microbiota and faecal metabolomics were used to characterise the host–microbe interface. Mechanistic studies were conducted in mouse colonoids, IBD biopsies and patient serum samples. Results DUOX2 activity increased permeability and bacterial translocation and induced subclinical inflammation in vTLR4 mice. GF vTLR4 mice had increased DUOX2 activity and permeability but no subclinical inflammation. In patients with IBD, DUOX2 expression was positively associated with plasma zonulin levels and negatively associated with ZO-1 expression. Engraftment of GF mice with IBD stool increased DUOX2 activity and triggered low-grade inflammation and permeability defects in mice. DUOX2 activity functionally altered the microbiome, reduced butyrate metabolism and promoted proinflammatory and pro-oncogenic bacterial metabolites. Butyrate and histone deacetylase (HDAC) inhibitors blocked DUOX2 activation and reversed its effects. Conclusions Elevated DUOX2 signalling contributes to epithelial barrier dysfunction, microbiome alterations and subclinical inflammation. Butyrate and HDAC inhibitors reversed these effects, indicating that DUOX2 may be a therapeutic target in IBD.

Human erythropoietin is mainly recognized for its hematopoietic function; however, by binding to its receptor (EpoR), it can activate different signaling pathways as STAT, PI3K, MAPK and RAS to increase cellular differentiation or provide neuroprotective effects, among others. A recombinant human erythropoietin variant with low glycosylation and without hematopoietic effect (EpoL) was purified from skimmed goat milk. Recombinant human erythropoietin (Epo) was obtained from CHO cell line and used as control to compare EpoL effects. Neuroprotection studies were performed in PC12 cells and rat hippocampal slices. Cells were pretreated during 1h with EpoL or Epo and exposed to oxidative agents (H₂O₂ or FCCP); cell viability was assayed at the end of the experiment by the MTT method. Hippocampal slices were exposed to 15min of oxygen and glucose deprivation (OGD) and the neuroprotective drugs EpoL or Epo were incubated for 2h post-OGD in re-oxygenated medium. Cell cultures stressed with oxidative agents, and pretreated with EpoL, showed neuroprotective effects of 30% at a concentration 10 times lower than that of Epo. Moreover, similar differences were observed in OGD ex vivo assays. Neuroprotection elicited by EpoL was lost when an antibody against EpoR was present, indicating that its effect is EpoR-dependent. In conclusion, our results suggest that EpoL has a more potent neuroprotective profile than Epo against oxidative stress, mediated by activation of EpoR, thus EpoL represents an important target to develop a potential biopharmaceutical to treat different central nervous system pathologies related to oxidative stress such as stroke or neurodegenerative diseases.

Multivalent live-attenuated or inactivated vaccines are often used to control the bovine viral diarrhea disease (BVD). Still, they retain inherent disadvantages and do not provide the expected protection. This study developed a new vaccine prototype, including the external segment of the E2 viral protein from five different subgenotypes selected after a massive screening. The E2 proteins of every subgenotype (1aE2, 1bE2, 1cE2, 1dE2, and 1eE2) were produced in mammalian cells and purified by IMAC. An equimolar mixture of E2 proteins formulated in an oil-in-water adjuvant made up the vaccine candidate, inducing a high humoral response at 50, 100, and 150 µg doses in sheep. A similar immune response was observed in bovines at 50 µg. The cellular response showed a significant increase in the transcript levels of relevant Th1 cytokines, while those corresponding to the Th2 cytokine IL-4 and the negative control were similar. High levels of neutralizing antibodies against the subgenotype BVDV1a demonstrated the effectiveness of our vaccine candidate, similar to that observed in the sera of animals vaccinated with the commercial vaccine. These results suggest that our vaccine prototype could become an effective recombinant vaccine against the BVD.