Outer Membrane Vesicles of a Human Commensal Mediate Immune Regulation and Disease Protection

Commensal bacteria impact host health and immunity through various mechanisms, including the production of immunomodulatory molecules. Bacteroides fragilis produces a capsular polysaccharide (PSA), which induces regulatory T cells and mucosal tolerance. However, unlike pathogens, which employ secretion systems, the mechanisms by which commensal bacteria deliver molecules to the host remain unknown. We reveal that Bacteroides fragilis releases PSA in outer membrane vesicles (OMVs) that induce immunomodulatory effects and prevent experimental colitis. Dendritic cells (DCs) sense OMV-associated PSA through TLR2, resulting in enhanced regulatory T cells and anti-inflammatory cytokine production. OMV-induced signaling in DCs requires growth arrest and DNA-damage-inducible protein (Gadd45α). DCs treated with PSA-containing OMVs prevent experimental colitis, whereas Gadd45α^(−/−) DCs are unable to promote regulatory T cell responses or suppress proinflammatory cytokine production and host pathology. These findings demonstrate that OMV-mediated delivery of a commensal molecule prevents disease, uncovering a mechanism of interkingdom communication between the microbiota and mammals

Dendritic Cells/Natural Killer Cross-Talk: A Novel Target for Human Immunodeficiency Virus Type-1 Protease Inhibitors

BACKGROUND: HIV-1 Protease Inhibitors, namely PIs, originally designed to inhibit HIV-1 aspartic protease, can modulate the immune response by mechanisms largely unknown, and independent from their activity on viral replication. Here, we analyzed the ability of PIs to interfere with differentiation program of monocytes toward dendritic cell (DCs) lineage, a key process in the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Monocytes from healthy donors were isolated and induced to differentiate in vitro in the presence or absence of saquinavir, ritonavir, nelfinavir, indinavir or amprenavir (sqv, rtv, nlfv, idv, apv, respectively). These drugs demonstrated a differential ability to sustain the generation of immature DCs (iDCs) with an altered phenotype, including low levels of CD1a, CD86, CD36 and CD209. DCs generated in the presence of rtv also failed to acquire the typical phenotype of mature DCs (mDCs), and secreted lower amounts of IL-12 and IL-15. Accordingly, these aberrant mDCs failed to support activation of autologous Natural Killer (NK) cells, and resulted highly susceptible to NK cell-mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: Our findings uncover novel functional properties of PIs within the DC-NK cell cross-talk, unveiling the heterogeneous ability of members of this class drugs to drive the generation of atypical monocyte-derived DCs (MDDCs) showing an aberrant phenotype, a failure to respond appropriately to bacterial endotoxin, a weak ability to prime autologous NK cells, and a high susceptibility to NK cell killing. These unexpected properties might contribute to limit inflammation and viral spreading in HIV-1 infected patients under PIs treatment, and open novel therapeutical perspectives for this class drugs as immunomodulators in autoimmunity and cancer

c-IAP1 and c-IAP2 are only partially redundant in T cells.

<p>(A) Purified T cells were cultured in vitro with the indicated amount of anti-CD3 in the absence (left panel) or presence (right panel) of 2 µg/ml of anti-CD28. After 48 hr, cells were pulsed with ³H-thymidine and harvested 18 hr later. The panel represents the average of four independent experiments and the error bars are the SEM. ***<i>p</i><0.005. (B) Expression of p100 and p52 in purified T cells was detected by immunoblotting. For each sample densitometry of p100 and p52 was performed and the results expressed as the ratio between each protein and β-actin. Expression of NIK (C) and c-IAP1/2 (D) in purified T cells was detected by immunoblotting. Values in C represent the ratio between NIK and β-actin. (E) p100/52 immunoblot of T cells freshly purified or stimulated for 24 hr with 1 µg/ml anti-CD3±2 µg/ml anti-CD28. (F) T cells were stimulated as in (E) and ELISA was performed on supernatants collected after 24 hr. One of two independent experiments is shown and the error bars are the SD of the duplicates. *<i>p</i><0.05.</p

c-IAP2 compensates for c-IAP1^H582A in non-canonical NF-κB inhibitory- and TNFR1 complexes.

<p>(A) Expression of p100 and p52 in MEFs was detected by immunoblotting. (B and F) MEFs were untreated (−) or transfected with non-targeting control (ctr) or c-IAP2 siRNA (c2) for 24 hr and lysed. p52 and c-IAP1/2 levels were determined by immunoblotting. IκBα expression was analyzed by immunoblotting of lysates of MEFs treated for the indicated times with 1 ng/ml of TNFα (C) or 15′ with the indicated doses of TNFα (D). (F) MEFs were untreated (-) or transfected with non-targeting control (ctr) or c-IAP2 siRNA for 24 hr, then treated with 1 ng/ml TNFα for 15′ and lysed. IκBα expression was analyzed by immunoblotting. Lanes were rearranged for clarity.</p

Generation of c-IAP1^H582A mice.

<p>(A) Schematic representation of the c-IAP1 targeting construct and the recombination strategy. (B) c-IAP1^H582A mice were distinguished from WT littermates by LR-PCR and Spe I digestion. (C) c-IAP1 and c-IAP2 expression in splenocytes from WT, c-IAP1^H582A heterozygous and homozygous mice were determined by immunoblotting. (D) <i>Ciap1</i> and <i>Ciap2</i> mRNA expression was determined in WT and c-IAP1^H582A splenocytes by real-time PCR. Bars represent the fold increase over WT expression. (E) WT or c-IAP1^H582A splenocytes were lysed in sample buffer after 8 hr of incubation with 25 mM chloroquine (CHQ), 1∶200 of the Sigma protease inhibitor cocktail (PIs), 10 µM MG-132, or complete medium (−) and c-IAP1 expression was analyzed by immunoblotting.</p