Antigen Presentation Capacity and Cytokine Production by Murine Splenic Dendritic Cell Subsets upon Salmonella Encounter

Abstract

Salmonella typhimurium is an intracellular bacterium that replicates in the spleen and mesenteric lymph nodes (MLN) of orally infected mice. However, little is known about the Ag presentation and cytokine production capacity of dendritic cells (DC), particularly CD8α+, CD8α−CD4−, and CD8α−CD4+ DC, from these organs in response to Salmonella. Infection of purified splenic DC with S. typhimiurium expressing green fluorescent protein (GFP) and OVA revealed that all three splenic DC subsets internalize bacteria, and splenic as well as MLN DC process Salmonella for peptide presentation. Furthermore, presentation of Salmonella Ags on MHC-I and MHC-II was evident in both CD8α+ and CD8α− splenic DC subsets. Direct ex vivo analysis of splenic DC from mice infected with GFP-expressing Salmonella showed that all three subsets harbored bacteria, and splenic DC purified from mice given Salmonella-expressing OVA presented OVA-derived peptides on MHC-I and MHC-II. Cytokine production analyzed by intracellular staining of splenic DC infected with GFP-expressing Salmonella revealed that TNF-α was produced by a large percentage of CD8α− DC, while only a minor proportion of CD8α+ DC produced this cytokine following bacterial exposure. In contrast, the greatest number of IL-12p40-producing DC were among CD8α+ DC. Experiments inhibiting bacterial uptake by cytochalasin D as well as use of a Transwell system revealed that bacterial contact, but not internalization, was required for cytokine production. Thus, DC in sites of Salmonella replication and T cell activation, spleen and MLN, respond to bacterial encounter by Ag presentation and produce cytokines in a subset-specific fashion.

CD8– dendritic cells preferentially cross‐present Saccharomyces cerevisiae antigens

“…Previously, Salmonella antigens have been shown to be cross-presented by both CD8 -DC and CD8 + DC [51]. Both pathogens are phagocytosed and killed by macrophages and neutrophils or evade the immune system by avoiding lysosomal degradation [52].Our data suggest that extracellular pathogens that are located in the phagosomes may preferentially be cross-presented by CD8 -DC to CD8 + T cells.…”

TNF-α-Dependent and -Independent Maturation of Dendritic Cells and Recruited CD11cintCD11b+ Cells during Oral Salmonella Infection

“…The inability of CD8␣ ϩ DC in PP to associate with Salmonella after oral infection is not due to an inability of this subset to phagocytose the bacteria, because both CD8␣ ϩ and CD8␣ Ϫ DC associate with Salmonella after i.v. infection (16,17). It is possible that the location of CD8␣…”

“…The influence of direct bacterial association on DC cytokine production during in vivo Salmonella infection is presently not known. However, in vitro, only a fraction of Salmonella-containing DC produces TNF-␣ or IL-12, and DC not directly associated with bacteria contribute substantially to cytokine production (16,17).…”

“…In addition, both CD8␣ ϩ and CD8␣ Ϫ DC of spleen and liver associate with Salmonella after i.v. infection, and CD11c ϩ DC from these organs can present Salmonella Ag ex vivo (16,17). It is not known where initial Ag presentation and T cell activation occur in vivo.…”

“…Before staining, samples were incubated with anti-Fc␥RII/III mAb (2.4G2; purified as described in Ref. 16) for 15 min at 4°C. Cells were then stained with FITC, PE, allophycocyanin, PE-Cy7, allophycocyanin-Cy7, or biotinylated conjugates of anti-CD11c (HL3), CD8␣ (53-6.7), CD80 (16-10A1), CD86 (GL1), CD40 (3/23), CD11b (M1/70), Gr-1 (RB6-8C5), B220 (RA3-6B2), MHC-II (M5/114.15.2), Mac-3 (M3/84), Ly-6C (AL-21), DEC-205 (NLDC-145), or F4/80 for 20 min at 4°C.…”