Salmonella enterica Serovar Typhimurium infection of dendritic cells leads to functionally increased expression of the macrophage-derived chemokine

Abstract

Gene expression in murine dendritic cells (DCs) infected with green fluorescent protein-expressing Salmonella enterica serovar Typhimurium BRD509 was studied by mRNA differential display. Infected DCs were sorted from uninfected cells by flow cytometry. The mRNA expression patterns of infected and uninfected cells revealed a number of differentially expressed transcripts, which included the macrophage-derived chemokine (MDC). Up-regulation of MDC transcription in infected DCs was confirmed by Northern blotting, and the kinetics of MDC expression was examined by real-time reverse transcription-PCR, with which 31- and 150-fold increases were detected at 2 and 6 h postinfection, respectively. The increased release by DCs of MDC into culture media was detected by an enzyme-linked immunosorbent assay. The biological activity of MDC was investigated in in vitro and in vivo assays. In vitro, supernatants from S. enterica serovar Typhimurium-infected DCs were chemoattractive to T cells, and neutralization of MDC in these supernatants inhibited T-cell migration. Passive transfer of anti-MDC antibody to mice infected with BRD509 revealed that neither growth of the bacterium nor resistance of the mice to reinfection was affected and that in vivo inhibition of MDC did not affect T-cell responses, as measured by the gamma interferon ELISPOT method 3 days after challenge infection.

FIG. 1.

Flow cytometric cell sorting of DCs after S. enterica serovar Typhimurium infection. DCs were either mock infected (A) or infected with GFP-expressing S. enterica serovar Typhimurium strain BRD509/GF3 for 6 h (B). Uninfected DCs and Salmonella-infected DCs were sorted with a flow cytometric cell sorter by using the phenotypes MHC-II ^low (box in panel A) and GFP ⁺ MHC-II ^low (box in panel B), respectively.

FIG. 2.

Up-regulation of MDC in DCs during S. enterica serovar Typhimurium infection. (A) Differential display was used to identify differentially expressed genes in DCs following S. enterica serovar Typhimurium BRD509/GF3 infection. A portion of a differential display gel is shown. The arrow indicates the position of a differentially displayed band whose intensity was reduced in uninfected DCs (lane U) compared with infected DCs (lane I). Subsequent sequencing and a BLAST search against the GenBank database assigned this differential display product to the MDC gene. (B) Northern blotting was used to confirm the increased expression of MDC. RNA was extracted from sorted uninfected and infected DCs, hybridized with the MDC-specific DNA probe, and then washed and rehybridized with the β-actin gene probe. The hybridization pattern was analyzed with a phosphorimager.

FIG. 3.

MDC expression in DCs. (A) Real-time RT-PCR was used to study the kinetic expression of the MDC gene in DCs following S. enterica serovar Typhimurium infection and sorting of infected and uninfected cells. The data are the relative expression levels of MDC compared to the expression levels of GAPDH in uninfected DCs which were sorted 2 h after mock infection (solid bar) or in S. enterica serovar Typhimurium-infected DCs sorted at different times postinfection (in hours) (cross-hatched bars). The results of one of two experiments are shown, and the results were similar in the different experiments. (B) MDC protein expression as determined by an ELISA (detection limit, <0.04 ng/ml). At 6 and 24 h after infection and sorting, culture supernatants from uninfected DCs (solid bars) and infected DCs (cross-hatched bars) were collected, and the amounts of MDC in the supernatants were analyzed. U6 and U24, uninfected cells at 6 and 24 h postinfection, respectively; I6 and I24, infected cells at 6 and 24 h postinfection, respectively.

FIG. 4.

MDC-induced chemotaxis of CD4 ⁺ T cells. Purified CD4 ⁺ cells (1.5 × 10 ⁵ cells) were added to the upper wells of a microchamber, and medium was added to the lower chamber. The data are the means and standard errors for duplicate samples. (A) Migratory responsiveness of CD4 ⁺ T cells to increasing doses of rmMDC (□) and in the presence of 10 μg of rat anti-mouse MDC antibody per ml (•). (B) Migration of CD4 ⁺ T cells induced by MDC in the DC culture supernatant. Bar 1, infected DC supernatant diluted 1:5; bar 2, infected DC supernatant diluted 1:5 plus anti-mouse MDC; bar 3, uninfected DC supernatant diluted 1:5; bar 4, uninfected DC supernatant diluted 1:5 plus anti-mouse MDC. (C) Migration of CD4 ⁺ T cells was inhibited by rabbit anti-MDC antiserum but not by the control antiserum. Bar 1, 250 ng of rmMDC per ml; bar 2, rmMDC plus anti-MDC antiserum diluted 1:5; bar 3, rmMDC plus anti-MDC antiserum diluted 1:50; bar 4, rmMDC plus control antiserum diluted 1:5; bar lane 5, rmMDC plus control antiserum diluted 1:50. (D) Bar 1, infected DC supernatant diluted 1:5; bar 2, infected DC supernatant diluted 1:5 plus anti-mouse MDC; bar 3, infected DC supernatant diluted 1:5 plus anti-MDC antiserum diluted 1:10; bar 4, infected DC supernatant diluted 1:5 plus control anti-GST antiserum diluted 1:10. The dotted lines indicate the background migration observed in medium-only control samples.

FIG. 5.

Effects of MDC-specific antiserum on in vivo anti-S. enterica serovar Typhimurium immune responses. Groups of five mice were injected i.p. with 0.5 ml of rabbit anti-MDC antiserum (cross-hatched bars) or isotype control anti-GST antiserum (open bars). The bars and error bars indicate the means and standard deviations, respectively, for each group of mice, and the solid dots indicate the values for individual mice. (A) On day zero, mice were injected i.p. with antiserum, and on day 1 mice were immunized intravenously with 100 CFU of S. enterica serovar Typhimurium BRD509. On days 7 and 14, two more injections of antiserum were administered. The bacterial load in each mouse spleen was determined by plating serial dilutions of spleen homogenates. (B) Naïve and BRD509-immunized mice were injected i.p. with antiserum 1 day prior to oral challenge infection with wild-type S. enterica serovar Typhimurium SL1344. On day 5 after the challenge, the bacterial load in each mouse spleen was determined by viability counting. (C) Effect of MDC neutralization on the number of IFN-γ-secreting cells present in the spleens of BRD509-immunized mice 3 days after challenge infection with SL1344 as determined by an ELISPOT assay. The nonparametric Mann-Whitney U test was used for statistical analysis.