Alarmin-loaded extracellular lipid droplets induce airway neutrophil infiltration during type 2 inflammation.

Group 2 innate lymphoid cells (ILC2s) play a crucial role in allergic diseases by coordinating a complex network of various effector cell lineages involved in type 2 inflammation. However, their function in regulating airway neutrophil infiltration, a deleterious symptom of severe asthma, remains unknown. Here, we observed ILC2-dependent neutrophil accumulation in the bronchoalveolar lavage fluid (BALF) of allergic mouse models. Chromatography followed by proteomics analysis identified the alarmin high mobility group box-1 (HMGB1) in the supernatant of lung ILC2s initiated neutrophil chemotaxis. Genetic perturbation of Hmgb1 in ILC2s reduced BALF neutrophil numbers and alleviated airway inflammation. HMGB1 was loaded onto the membrane of lipid droplets (LDs) released from activated lung ILC2s. Genetic inhibition of LD accumulation in ILC2s significantly decreased extracellular HMGB1 abundance and BALF neutrophil infiltration. These findings unveil a previously uncharacterized extracellular LD-mediated immune signaling delivery pathway by which ILC2s regulate airway neutrophil infiltration during allergic inflammation.

Retinoid X receptor gamma dictates the activation threshold of group 2 innate lymphoid cells and limits type 2 inflammation in the small intestine.

Group 2 innate lymphoid cells (ILC2s) are crucial in promoting type 2 inflammation that contributes to both anti-parasite immunity and allergic diseases. However, the molecular checkpoints in ILC2s that determine whether to immediately launch a proinflammatory response are unknown. Here, we found that retinoid X receptor gamma (Rxrg) was highly expressed in small intestinal ILC2s and rapidly suppressed by alarmin cytokines. Genetic deletion of Rxrg did not impact ILC2 development but facilitated ILC2 responses and the tissue inflammation induced by alarmins. Mechanistically, RXRγ maintained the expression of its target genes that support intracellular cholesterol efflux, which in turn reduce ILC2 proliferation. Furthermore, RXRγ expression prevented ILC2 response to mild stimulations, including low doses of alarmin cytokine and mechanical skin injury. Together, we propose that RXRγ expression and its mediated lipid metabolic states function as a cell-intrinsic checkpoint that confers the threshold of ILC2 activation in the small intestine.

Vitamin D receptor enhances NLRC4 inflammasome activation by promoting NAIPs-NLRC4 association.

Inflammasomes are cytosolic multiprotein complexes that initiate host defense against bacterial pathogens. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family caspase-associated recruitment domain-containing protein 4 (NLRC4) inflammasomes plays a critical role in the inflammatory response against intracellular bacterial infection. The NLR family apoptosis inhibitory proteins (NAIPs) detect Flagellin or type III secretion system (T3SS) microbial components to activate NLRC4 inflammasome. However, the underlying mechanism of NLRC4 inflammasome activation is not completely understood. Here, we show that the vitamin D receptor (VDR) is an essential immunological regulator of the NLRC4 inflammasome. Conditional VDR knockout mice (VDRflox/flox lyz2-Cre) exhibited impaired clearance of pathogens after acute Salmonella Typhimurium infection leading to poor survival. In macrophages, VDR deficiency reduced caspase-1 activation and IL-1ß secretion upon S. Typhimurium infection. For NAIPs act as upstream sensors for NLRC4 inflammasome assembly, the further study demonstrated that VDR promoted the NAIP-NLRC4 association and triggered NAIP-NLRC4 inflammasome activation, not NLRP3 activation. Moreover, Lys123 residue of VDR is identified as the critical amino acid for VDR-NLRC4 interaction, and the mutant VDR (K123A) effectively attenuates the NLRC4 inflammasome activation. Together, our findings suggest that VDR is a critical regulator of NAIPs-NLRC4 inflammasome activation, mediating innate immunity against bacterial infection.

Parkin increases the risk of colitis by downregulation of VDR via autophagy-lysosome degradation.

Parkin, an E3 ubiquitin ligase, plays an essential role in mitophagy. Emerging evidence indicates that mitophagy is involved in various processes closely related to immune diseases, including inflammatory bowel diseases (IBD). Here, the authors show that Parkin increases the occurrence of colitis and severe inflammation. Deletion of Parkin resulted in marked reductions in colonic inflammation and exhibited high resistance to DSS-induced colitis. Mechanism investigation indicated that Parkin interacts with Vitamin D receptors (VDR), a critical inhibitory regulator in IBD. Parkin promotes VDR degradation via the p62-related autophagy-lysosome pathway. Comparison of colitis in Parkin-/- and Parkin-/-Vdr-/- mice showed that the protective effect of Parkin deletion against colitis was abolished by VDR deletion. The result suggests that the regulatory effect of Parkin in colitis is a VDR-dependent pathway. Our research provides a new role of Parkin in colitis by downregulating VDR, which provides a potential strategy for treating IBD.

Effects of Dietary Protein Levels on Fecal Amino Acids Excretion and Apparent Digestibility, and Fecal and Ileal Microbial Amino Acids Composition in Weaned Piglets.

Background and Aims: The purpose of this study was to determine the effects of low protein diets with the same Lys, Met + Cys, Thr, and Trp levels as in high protein diets on the fecal amino acid excretion and apparent digestibility, and ileal and fecal microbial amino acids composition in weaned piglets. Methods: Fifty-four 21-day-old Duroc × Landrace × Yorkshire weaned piglets were randomly divided into three groups and fed with corn-soybean meal basal diets, in which the crude protein (CP) content was 20% (H-CP), 17% (M-CP), and 14% (L-CP), respectively. The experiment included a 7-day adaptation period and a 45-day trial period. Six piglets in each group were randomly slaughtered on days 10, 25, and 45 of the trial period, and the intestinal contents, intestinal mucosa, and feces were collected. Results: The results showed that the interaction between feeding time and dietary CP levels was reflected in the apparent digestibility of dietary CP and amino acid (AA) (p < 0.01). With the increase of age, the apparent digestibility of CP and AA were increased (p < 0.01). With the increase of CP levels, the excretion of nitrogen (N) was decreased (p < 0.01), whereas the flow of microbial AA in the ileum and feces were increased (p < 0.01). The interaction between feeding time and dietary CP levels was also reflected in the composition of AA in the ileum and stool of piglets (p < 0.01). The proportion of His, Lyr, Met, Cys, and Ser was lower than the average, whereas the proportion of Phe, Leu, Pro, Ala, Glu, and Asp was higher than the average. With the increase of age, the AA content of microorganisms increased (p < 0.01). Conclusion: All in all, this work revealed the changes of N, CP, and AA excretion and digestibility of feces and microorganisms of piglets under the combined action of different dietary protein levels and different feeding times, and also the changes of AA composition of intestinal microorganisms and AA composition of microorganisms.

Vitamin D Receptor Inhibits NLRP3 Activation by Impeding Its BRCC3-Mediated Deubiquitination.

The NLRP3 inflammasome is a multiprotein oligomer responsible for activation of the inflammatory response by promoting the maturation and secretion of the pro-inflammatory cytokines IL-1ß and IL-18. Dysregulation of this inflammasome has been linked to several autoimmune diseases, indicating that NLRP3 is tightly regulated to prevent aberrant activation. The regulation of NLRP3 activation remains unclear. Here, we report the identification of vitamin D receptor (VDR) as a negative regulator of NLRP3 oligomerization and activation. VDR can physically bind NLRP3 and block the association of NLRP3 with BRCC3. When BRCC3-mediated deubiquitination of NLRP3 is inhibited by VDR, NLRP3 activation is subsequently inhibited. In the absence of VDR, caspase-1 activation and IL-1ß release are increased in response to LPS-induced inflammation or alum-induced peritoneal inflammation, indicating that VDR is a negative regulator of NLRP3 inflammasome activation in vivo. In addition, vitamin D negatively regulates the NLRP3 inflammasome via VDR signaling to effectively inhibit IL-1ß secretion. These studies demonstrate that VDR signaling constrains NLRP3 inflammasome activation and might be a potential treatment target for NLRP3 inflammasome-related diseases.

Corrigendum: 1,25-Dihydroxyvitamin D Inhibits LPS-Induced High-Mobility Group Box 1 (HMGB1) Secretion via Targeting the NF-E2-Related Factor 2-Hemeoxygenase-1-HMGB1 Pathway in Macrophages.

[This corrects the article on p. 1308 in vol. 8, PMID: 29085368.].

Sphingosine kinase 2 cooperating with Fyn promotes kidney fibroblast activation and fibrosis via STAT3 and AKT.

Sphingosine kinases (Sphks) are the rate-limiting enzymes in the conversion of sphingosine to biologically active sphingosine-1-phosphate. The present study aimed to determine the role of Sphk2 and its downstream targets in renal fibroblast activation and interstitial fibrosis. In the kidney interstitium of patients with renal fibrosis, Sphk2high-expressing cells (mainly interstitial fibroblasts) were significantly elevated and highly correlated with disease progression in patients. In a murine model of renal interstitial fibrosis, Sphk2 was upregulated in the kidney of wild-type mice in response to disease progression. Importantly, Sphk2-knockout (KO) mice exhibited significantly lower levels of extracellular matrix (ECM) production and a suppressed inflammatory response in the kidney tissues, compared to those in their wild-type counterparts, whereas the expression of TGF-ß1 was unaffected. TGF-ß1 effectively upregulated Sphk2 expression in the renal interstitial fibroblast line, NRK-49F, independent of canonical Smad signaling activation. Furthermore, siRNA-mediated Sphk2 knockdown or suppression of Sphk2 activity by ABC294640 exposure effectively attenuated AKT and STAT3 activation and ECM production, but had no effects on Smad2 and Smad3 activation. Sphk2 phosphorylated Fyn to activate downstream STAT3 and AKT, thereby promoting ECM synthesis. Therefore, our findings indicate that targeting Sphk2-Fyn-STAT3/AKT signaling pathway may be a novel therapeutic approach for renal fibrosis.

1,25-Dihydroxyvitamin D Inhibits LPS-Induced High-Mobility Group Box 1 (HMGB1) Secretion via Targeting the NF-E2-Related Factor 2-Hemeoxygenase-1-HMGB1 Pathway in Macrophages.

1,25-Dihydroxyvitamin D [1,25(OH)2D3] is recognized as a key mediator of inflammatory diseases, including sepsis. Clinical studies demonstrate that 1,25 (OH)2D3 protects patients from sepsis, but clinical treatment with 1,25(OH)2D3 is rare. In this study, we report that 1,25(OH)2D3 treatment has beneficial effects and improves the survival rate in LPS-induced mouse sepsis model by blocking the secretion of high-mobility group box 1 (HMGB1), a key late regulator of sepsis. LPS-induced HMGB1 secretion is attenuated by 1,25(OH)2D3via blocking HMGB1 translocation from the nucleus to the cytoplasm in macrophages. 1,25(OH)2D3 can induce the expression of hemeoxygenase-1 (HO-1), which is essential for blocking HMBG1 nuclear translocation and its secretion. When siHO-1 or an HO-1 inhibitor are used, the effect of 1,25(OH)2D3 on inhibition of HMGB1 secretion is suppressed. Considering that HO-1 is a downstream gene of NF-E2-related factor 2 (Nrf2), we further confirm that Nrf2 activation can be activated by 1,25(OH)2D3 upon LPS exposure. Together, we provide evidence that 1,25(OH)2D3 attenuates LPS-induced HMGB1 secretion via the Nrf2/HO-1 pathway in macrophages.