Activation-Induced TIM-4 Expression Identifies Differential Responsiveness of Intestinal CD103+ CD11b+ Dendritic Cells to a Mucosal Adjuvant.

Macrophage and dendritic cell (DC) populations residing in the intestinal lamina propria (LP) are highly heterogeneous and have disparate yet collaborative roles in the promotion of adaptive immune responses towards intestinal antigen. Under steady-state conditions, macrophages are efficient at acquiring antigen but are non-migratory. In comparison, intestinal DC are inefficient at antigen uptake but migrate to the mesenteric lymph nodes (mLN) where they present antigen to T cells. Whether such distinction in the roles of DC and macrophages in the uptake and transport of antigen is maintained under immunostimulatory conditions is less clear. Here we show that the scavenger and phosphatidylserine receptor T cell Immunoglobulin and Mucin (TIM)-4 is expressed by the majority of LP macrophages at steady-state, whereas DC are TIM-4 negative. Oral treatment with the mucosal adjuvant cholera toxin (CT) induces expression of TIM-4 on a proportion of CD103+ CD11b+ DC in the LP. TIM-4+ DC selectively express high levels of co-stimulatory molecules after CT treatment and are detected in the mLN a short time after appearing in the LP. Importantly, intestinal macrophages and DC expressing TIM-4 are more efficient than their TIM-4 negative counterparts at taking up apoptotic cells and soluble antigen ex vivo. Taken together, our results show that CT induces phenotypic changes to migratory intestinal DC that may impact their ability to take up local antigens and in turn promote the priming of mucosal immunity

CD103+ CD11b+ TIM-4+ DC are enriched in the mLN of CT treated mice suggesting migration from the LP.

<p>(A) Cells isolated from the mLN were assessed for expression of CFSE 17 hours after oral administration of CT and CFDA-SE. (B) mLN DC subset frequencies from untreated (NT) and CT-treated mice were identified by flow cytometry and used to calculate cell numbers. Bar graph shows average total number of CD11c+ MHCII^hi DC in the mLN ±SEM for 9 mice from 3 independent experiments with 3 mice per group. Statistical significance was determined by Student’s unpaired t-test. *** 0.00010.05, * p <0.05, ** 0.001</p

TIM-4 expression is associated with enhanced endocytic ability in APC from the SI.

<p>Cells were isolated from the SI LP of CT treated mice and enriched for CD11c+ cells by magnetic selection. (A) CD11c+ cells were co-cultured with e670 labelled (AB-e670) or unlabelled apoptotic bodies (AB) for 1 hour and TIM-4+ and TIM-4− macrophages assessed for e670 fluorescence by flow cytometry. (B) As in A, except TIM-4^hi, TIM-4^int and TIM-4− DC were assessed for e670 fluorescence. (C) CD11c+ cells were co-cultured with ovalbumin (OVA)-AF488 or OVA for 1 hour and macrophages assessed for AF488 fluorescence by flow cytometry. (D) As in C, except TIM-4^hi, TIM-4^int and TIM-4− DC were assessed for AF488 fluorescence. Histograms show data representative of 3–5 independent experiments and bar graphs show median fluorescent intensity (MFI)±SEM of data pooled from 3–5 independent experiments. Statistical significance was determined by one-way ANOVA with Bonferroni post-test. n.s. p >0.05, * p <0.05, ** 0.0010.05, * p <0.05, ** 0.001</p

CD103+ CD11b+ TIM-4+ DC in the SI LP express increased levels of co-stimulatory molecules compared to TIM-4− DC.

<p>(A) SI DC and macrophage subsets from untreated (NT) and CT treated mice were identified using flow cytometry and compared for expression of CD86. (B) CD103+ CD11b+ DC from CT treated mice were divided into TIM-4−, TIM-4^int and TIM-4^hi subsets and compared to untreated CD103+ CD11b+ TIM-4− DC for expression of CD86. (C) As in B, except that CD40, CD80, PDL-1 and PDL-2 expression was compared. Bar graphs show average median fluorescence intensity (MFI)±SEM for 3 mice. Statistical significance was determined by one-way ANOVA with Bonferroni post-test. n.s. p >0.05, * p <0.05, ** 0.001</p

Macrophages, but not DC, from the SI LP express TIM-4 at steady-state.

<p>APC were isolated from the SI of untreated mice and analysed for expression of cell surface molecules by flow cytometry. (A) Dot plots show the gating strategy employed to identify distinct SI DC and macrophage (Mφ) populations. (B) Histograms compare expression profiles of cell surface molecules and RALDH between DC and macrophage populations identified as in (A). (C) Intestinal macrophages were divided into TIM-4− and TIM-4+ populations and compared for expression of F4/80 and CX3CR1. Bar graphs show average median fluorescent intensity (MFI)±SEM for 3 individual mice. Data are from 1 of 3 independent experiments, each with 3 mice, that gave similar results.</p

GPX4 regulates cellular necrosis and host resistance in Mycobacterium tuberculosis infection.

Cellular necrosis during Mycobacterium tuberculosis (Mtb) infection promotes both immunopathology and bacterial dissemination. Glutathione peroxidase-4 (Gpx4) is an enzyme that plays a critical role in preventing iron-dependent lipid peroxidation-mediated cell death (ferroptosis), a process previously implicated in the necrotic pathology seen in Mtb-infected mice. Here, we document altered GPX4 expression, glutathione levels, and lipid peroxidation in patients with active tuberculosis and assess the role of this pathway in mice genetically deficient in or overexpressing Gpx4. We found that Gpx4-deficient mice infected with Mtb display substantially increased lung necrosis and bacterial burdens, while transgenic mice overexpressing the enzyme show decreased bacterial loads and necrosis. Moreover, Gpx4-deficient macrophages exhibited enhanced necrosis upon Mtb infection in vitro, an outcome suppressed by the lipid peroxidation inhibitor, ferrostatin-1. These findings provide support for the role of ferroptosis in Mtb-induced necrosis and implicate the Gpx4/GSH axis as a target for host-directed therapy of tuberculosis

Homeostatic IL-13 in healthy skin directs dendritic cell differentiation to promote TH2 and inhibit TH17 cell polarization

The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.</p