Microbiome-derived metabolites influence intestinal homeostasis and regulate graft-versus-host disease (GVHD), but the molecular mechanisms remain unknown. Here we show the metabolite sensor G-protein-coupled receptor 43 (GPR43) is important for attenuation of gastrointestinal GVHD in multiple clinically relevant murine models. GPR43 is critical for the protective effects of short-chain fatty acids (SCFAs), butyrate and propionate. Increased severity of GVHD in the absence of GPR43 is not due to baseline differences in the endogenous microbiota of the hosts. We confirm the ability of microbiome-derived metabolites to reduce GVHD by several methods, including co-housing, antibiotic treatment, and administration of exogenous SCFAs. The GVHD protective effect of SCFAs requires GPR43-mediated ERK phosphorylation and activation of the NLRP3 inflammasome in non-hematopoietic target tissues of the host. These data provide insight into mechanisms of microbial metabolite-mediated protection of target tissues from the damage caused allogeneic T cells.

Cross-presentation initiates immune responses against tumors and viral infections by presenting extracellular antigen on MHC I to activate CD8+ T cell-mediated cytotoxicity. In vitro studies in dendritic cells (DCs) established SNARE protein SEC22B as a specific regulator of cross-presentation. However, the in vivo contribution of SEC22B to cross-presentation has not been tested. To address this, we generated DC-specific Sec22b knockout (CD11c-Cre Sec22bfl/fl) mice. Contrary to paradigm, SEC22B-deficient DCs efficiently cross-present both in vivo and in vitro. Though in vitro shRNA-mediated Sec22b silencing in bone marrow-derived dendritic cells (BMDCs) reduced cross-presentation, treatment of SEC22B-deficient BMDCs with the same shRNA produced a similar defect, suggesting the Sec22b shRNA modulates cross-presentation through off-target effects. RNAseq of Sec22b shRNA-treated SEC22B-deficient BMDCs demonstrated several changes in the transcriptome. Our data demonstrate that, contrary to the accepted model, that SEC22B is not necessary for cross-presentation, cautioning against extrapolating phenotypes from knockdown studies alone.

In the version of the article originally published, in the top immunoblot (loading control) in Figure 5f, the right half was incorrectly a mirrorimage duplication of the left half. The correct immunoblot from a replicate experiment is now presented (along with the corresponding bottom immunoblot). The error has been corrected in the HTML and PDF versions of the article.Corrigendum: Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease

The highly conserved SNARE protein SEC22B mediates diverse and critical functions, including phagocytosis, cell growth, autophagy, and protein secretion. However, these characterizations have thus far been limited to in vitro work. Here, we expand our understanding of the role Sec22b plays in vivo . We utilized Cre-Lox mice to delete Sec22b in three tissue compartments. With a germline deletion of Sec22b , we observed embryonic death at E8.5. Hematopoietic/endothelial cell deletion of Sec22b also resulted in in utero death. Notably, mice with Sec22b deletion in CD11c-expressing cells of the hematopoietic system survive to adulthood. These data demonstrate Sec22b contributes to early embryogenesis through activity both in hematopoietic/endothelial tissues as well as in other tissues yet to be defined.

The immune system regulates many processes vital to homeostasis. Antigen cross-presentation, an immune process specific to dendritic cells (DCs), critically contributes to maintaining homeostasis by regulating immune tolerance, antiviral activity, and antitumor responses. Through cross-presentation, extracellular antigen is processed and presented by MHC I on DCs to CD8 T cells. Despite cross-presentation's crucial role in mediating immune responses, its molecular regulation remains poorly defined. A potential breakthrough came when, using an in vitro shRNA-mediated knockdown (KD) approach, a group identified Sec22b as a central regulatory molecule of the pathway1. They demonstrated that Sec22b mediates recruitment of proteins necessary for MHC I-antigen loading from the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) to the antigen-containing phagosome, promoting cross-presentation. Thus, we tested the hypothesis that Sec22b mediates cross-presentation in vivo, in the context of an intact immune system under physiological conditions. We generated DC-specific Sec22b knockout (KO) mice (CD11cCre Sec22bfl/fl) from Sec22b-conditional gene trapped founder mice. These mice develop normally and have an intact immune system. KO DCs from spleen and bone marrow (BM) have Cre-mediated excision at the Sec22b locus, verified by PCR, and reduction of Sec22b production, verified by Western Blot. KO DCs from spleen and BM express activation markers in response to TLR and NLR stimulation at comparable levels to Cre- Sec22bfl/fl (FL) mice. By adoptively transferring ovalbumin (OVA)-specific (OT-I) T cells into these mice, then injecting with soluble OVA i.p., we measured OT-I T cell proliferation as a readout of cross-presentation. To our surprise, we saw no difference in the ability of KO versus FL mice to cross-present OVA (p >0.9). This observation was verified with in vitro assays with KO DCs from BM and spleen cross-presenting soluble OVA (0-3 mg/mL). We obtained similar findings using bead-bound, insoluble OVA. From this, we concluded Sec22b is not necessary for cross-presentation, invalidating our hypothesis. We were, however, able to reduce cross-presentation by shRNA-mediated KD of Sec22b (p <0.05), reproducing published observations. This discrepancy in observations was not due to functional compensation in KO BMDCs by Sec22b homologs, Sec22a or Sec22c, which we determined using qPCR. Intriguingly, when we treated KO BMDCs with the Sec22b-targeting shRNA, we again observed a reduction in cross-presentation (p <0.05). The reduction was comparable to that found in Sec22b-targeting shRNA-treated FL and WT BMDCs. Taken together, our data (a) demonstrate that Sec22b is not necessary for cross-presentation, (b) suggest the existence of a novel critical mediator of cross-presentation that is also targeted by the shRNA sequence used and (c) caution against extrapolating mechanisms or phenotypes based on KD studies alone. Cebrian, I. et al. Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells. Cell147, 1355-1368 (2011). Disclosures No relevant conflicts of interest to declare.

Crosspresentation critically contributes to the adaptive immune response to tumors and viral infection by presenting extracellular antigen on MHC I to activate CD8 T cells. However, its molecular regulation is poorly understood. Using in vitro shRNA-mediated knockdown (KD) methods, another group had shown that Sec22b, an ER-SNARE, specifically regulates crosspresentation. Thus, we tested the hypothesis that Sec22b would mediate crosspresentation in vivo. We generated DC-specific Sec22b knockout (KO) mice, which have an intact, functional immune system. After adoptively transferring OT-I T cells into these mice, then injecting with soluble OVA i.p., we analyzed OT-I T cell proliferation to measure crosspresentation. Surprisingly, we saw no difference in OVA crosspresentation by KO versus FL mice. This observation was verified in vitro using KO DCs from BM and spleen crosspresenting soluble and insoluble OVA. We concluded Sec22b is not necessary for crosspresentation, invalidating our hypothesis. However, we were able to reproduce the reduced crosspresentation by shRNA-mediated KD of Sec22b. Intriguingly, when we treated KO BMDCs with the same Sec22b-targeting shRNA, we again observed reduced crosspresentation comparable to that seen in shRNA-treated WT BMDCs. This suggested the shRNA mediates crosspresentation through its effects on an off-target gene. RNAseq data on shRNA-treated BMDCs provided a candidate list of potential crosspresentation mediators. Our data demonstrates that (a) Sec22b is not necessary for crosspresentation, (b) there may be a novel critical mediator of crosspresentation targeted by the shRNA, and (c) extrapolating mechanisms or phenotypes based on KD studies alone must be done with caution.