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

Abstract

Summary 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.

The essential role of complement in antibody‐mediated resistance to Salmonella

“…Opsonization was performed by incubating bacteria at ~10 8  CFU/ml with 6·75 μg/ml of anti‐O4 monoclonal IgG2a, or with isotype control IgG2a, at 37° with shaking for 30 min; non‐bound antibodies were removed by washing with PBS. The optimal concentration of anti‐O4 IgG2a for opsonizing bacteria was determined as the lowest dilution that would not cause bacterial agglutination 7. Effective opsonization was visualized with fluorescence microscopy using Alexa Fluor 568‐conjugated goat anti‐mouse IgG (Invitrogen, Paisley, UK).…”

“…In fact, protection against oral infection in immunized animals, which is IgG dependent,10 does not require the C3 component of complement, but is dependent on the function of Fc γ RI, II and III 4. Moreover, uptake and killing of IgG‐opsonized Salmonella by murine macrophages and human phagocytic cell lines can be mediated by Fc γ Rs in the absence of complement 5, 6, 7. However, poor complement activity has been inferred to be the cause of the poor antibody‐dependent bactericidal activity of mouse serum against non‐typhoidal strains of Salmonella 8.…”

Igg Subclasses Targeting the Flagella of Salmonella Enterica Serovar Typhimurium Can Mediate Phagocytosis and Bacterial Killing

“…Following opsonisation, the bacteria were added to the THP-1 cells at multiplicity of infection (MOI) of 10:1 (bacteria: THP-1 cells) and incubated for 45 min at 37 °C [39]. The infected cells were then washed five times with PBS.…”

“…Bacteria were opsonised as previously described [39]. Briefly, bacteria from an overnight culture were opsonised by incubation in either the humanised anti-TSSPSAD antibodies or the non-specific control antibody at a final concentration of 25 μg/ml (the highest concentration that does not agglutinate the bacteria) at 37°C with shaking for 30 min.…”

“…We have previously shown that targeting the surface-abundant OmpA protein with human IgGs can increase the uptake and killing of bacteria by human phagocytic cells, the efficiency of these functions depending both on the subclass of the IgG and the FcR that is available for antibody binding [39]. Broadening the repertoire of antigens to be included in future iNTS multicomponent vaccines is desirable.…”