Human IgG isotypes and activating Fcγ receptors in the interaction of Salmonella enterica serovar Typhimurium with phagocytic cells

Abstract

Several classes and multiple subclasses of immunoglobulins are produced towards protein and polysaccharide antigens in response to Salmonella infection and play a key role in protection against systemic disease. The targeting of Salmonella to Fc receptors (FcR) on phagocytes is a key step in the antibody-mediated antibacterial functions of host cells. We wished to compare the relative efficiency of different human IgG subclasses, which targeted the Salmonella enterica OmpA surface protein in modulating the interaction of bacteria with human phagocytes. To this end, we developed a novel system by tagging OmpA with a foreign CD52 mimotope (TSSPSAD) and opsonizing the bacteria with a panel of humanized CD52 antibodies that share the same antigen-binding V-region, but have constant regions of different subclasses. Our data revealed that opsonization with all the IgG subclasses increases Salmonella uptake by human phagocytes. IgG3 resulted in the highest level of bacterial uptake and the highest average bacterial load per infected cell, which was closely followed by IgG1, then IgG4 and lastly IgG2. Phagocytosis mediated by IgG1, IgG3 and IgG4 had a higher dependency on FcγRI than FcγRIIA, whereas IgG2-mediated phagocytosis required FcγRIIA more than FcγRI. The results show that IgG binding to OmpA increases the uptake of Salmonella by human phagocytic cells and that the efficiency of this process depends both on the subclass of the IgG and the type of FcR that is available for antibody binding.

Figure 1

Percentage of infected cells after phagocytosis of Salmonella Typhimurium opsonized with different human IgG subclasses. THP-1 cells were infected with S. Typhimurium opsonized with anti-TSSPSAD antibodies or with bacteria exposed to control IgG. The anti-TSSPSAD antibodies of different subclasses, either IgG1, IgG2, IgG3 or IgG4, are labelled as 1, 2, 3, and 4, respectively and the non-specific control antibody is labelled as N; 450 cells were counted for each of the three experimental repeats. The graph shows the percentages of cells harbouring visible intracellular bacteria which are expressed as the mean percentage of infected cells from three experimental repeats. The error bars represent standard deviations. Every possible paired combination of groups was considered for comparison (i.e. N versus 1, N versus 2, N versus 3, N versus 4, 1 versus 2, 1 versus 3, 1 versus 4, 2 versus 3, 2 versus 4, 3 versus 4) and all P values are < 0·01.

Figure 2

Number of intracellular bacteria per infected cell after phagocytosis of Salmonella Typhimurium opsonized with different human IgG subclasses. THP-1 cells were infected as described in Fig. 1; 450 cells were counted for each of the three experimental repeats. The graph shows the distributions of visible intracellular bacteria per infected cell, which are expressed as the mean distributions from three experimental repeats. Every possible paired combination of groups was considered for comparison (See Fig. 1 legend) and all P values are < 0·0013.

Figure 3

Viability of intracellular bacteria after phagocytosis of Salmonella Typhimurium opsonized with different human IgG subclasses. THP-1 cells were infected as described in Fig. 1. The graph shows the number of culturable intracellular bacteria (viable count) recovered from THP-1 cells. Data are expressed as the mean of three experimental repeats and the error bars represent standard deviations. Every possible paired combination of groups was considered for comparison (See Fig. 1 legend) and all P-values are < 0·0034.

Figure 4

Importance of Fcγ receptors (FcγR) in mediating the percentage of infected cells. THP-1 cells were pre-incubated with anti-FcγR antibodies before being exposed to IgG1- (a), IgG2- (b), IgG3- (c), IgG4- (d) opsonized bacteria. The percentage of infected cells was determined by fluorescence microscopy; 450 cells were counted for each of the three experiment repeats. Data are expressed as the mean percentage of infected cells from three experimental repeats and the error bars represent standard deviations. The percentage of infected cells observed when the activating FcγRs were blocked either individually or in combination was compared with the percentage of infected cells observed when none of the FcγRs were blocked. For each figure panel (a, b, c or d), all possible paired combinations of FcγR blocking, with the exception of FcγRIII blocking (alone and in combination), were considered for comparison. FcγRIII blocking was excluded as there was negligible effect of FcγRIII blocking on the percentage of infected cells. For the combinations considered (which exclude those combinations involving FcγRIII), the P-values are < 0·015 for IgG1 (a), < 0·048 for IgG2 (b), < 0·026 for IgG3 (c) and < 0·03 for IgG4 (d).

Figure 5

Importance of Fcγ receptors (FcγR) in affecting the bacterial load of infected cells. Cells were pre-incubated with anti-FcγR antibodies before being exposed to IgG1- (a), IgG2- (b), IgG3- (c), IgG4- (d) opsonized bacteria. The bacterial load (number of visible intracellular bacteria) per infected cell was determined by microscopy; 450 cells were counted for each of the three experiment repeats. Data were expressed as the mean distributions of visible intracellular bacteria per infected cell from three experimental repeats. The distributions of visible intracellular bacteria observed when the activating FcγRs were blocked either individually or in combination was compared with the distribution of visible intracellular bacteria observed when none of the FcγRs was blocked. For each figure panel (a, b, c or d), all possible paired combinations of FcγR blocking, with the exception of FcγRIII blocking (alone and in combination), were considered for comparison (See Fig. 4 legend). FcγRIII blocking was excluded as there was negligible effect of FcγRIII blocking on the bacterial load of infected cells. The P-values were < 0·0013 for IgG1 (a), 0·05 for IgG2 (b), 9·1 × 10⁻⁴ for IgG3 (c) and < 0·051 for IgG4 (d).

Figure 6

Importance of Fcγ receptors (FcγR) in affecting the intracellular viable bacterial count. Cells were pre-incubated with anti-FcγR antibodies and then exposed to IgG1- (a), IgG2- (b), IgG3- (c), IgG4- (d) opsonized bacteria. The intracellular viable bacteria were determined by pour plates. Data are expressed as the mean of three experimental repeats and the error bars represent standard deviations. The intracellular viable bacterial count observed when the activating FcγRs were blocked either individually or in combination was compared with the percentage of infected cells observed when none of the FcγRs was blocked. For each figure panel (a, b, c or d), every possible paired combination of FcγR blocking was considered, with the exception of FcγRIII blocking (alone and in combination). FcγRIII blocking was excluded as there was negligible effect of FcγRIII blocking on the intracellular viable bacterial count. The P-values are < 0·0049 for IgG1 (a), < 0·026 IgG2 (b), < 0·014I for gG₃ (c) and < 0·02 for IgG4 (d).