Polymorphonuclear cells (neutrophils) are the first cells that arrive at sites of infections. According to the current dogma, they are involved in eliminating bacteria, after which they die through apoptosis. We now demonstrate that enhanced IgA-induced phagocytosis of bacteria or beads by neutrophils led to increased cell death. Nuclear changes and positivity for the general cell death marker 7-aminoactinomycin D were observed, but the absence of annexin V membrane staining supported that neutrophils did not die via apoptosis, in contrast to neutrophils that had not phagocytosed bacteria. Moreover, increased release of neutrophil extracellular traps (NETs) was observed, which was most likely due to augmented production of reactive oxygen species after uptake of IgA-opsonized particles. Blocking the IgA Fc receptor FcαRI abrogated phagocytosis and NET formation. Thus, FcαRI triggering on neutrophils resulted in a rapid form of cell death that is referred to as NETosis, as it is accompanied by the release of NETs. As such, IgA may play a prominent role in mucosal inflammatory responses, where it is the most prominent Ab, because it enhanced both phagocytosis of bacteria and formation of NETs, which are effective mechanisms that neutrophils employ to eliminate pathogens.

Immunoglobulin A (IgA) is the most prevalent antibody at mucosal sites, and has an important role in defense by preventing invasion of pathogens. Traditionally, IgA has been thought of as a non-inflammatory antibody that helps to maintain homeostasis in the mucosa. However, in the last decade it has become clear that IgA is a very potent stimulus to initiate pro-inflammatory cellular processes, especially after triggering the IgA Fc receptor (FcαRI) on neutrophils. It was furthermore described that FcαRI acts as a regulator between anti- and pro-inflammatory responses of IgA. Although neutrophil activation is beneficial in (mucosal) infections, abnormal or excessive IgA immune complexes can induce disproportionate neutrophil migration and in this way initiate a perpetuating neutrophil recruitment and activation loop, which will result in severe tissue damage. Increasing evidence on this process plays a detrimental role in several diseases, including autoimmune IgA blistering diseases, a subtype of rheumatoid arthritis and ulcerative colitis. Inhibiting FcαRI-mediated activation may dampen inflammation in these patients. This process also opens up the possibility of targeting FcαRI in antibody immunotherapy of cancer. Thus, interfering with IgA-mediated FcαRI activation may represent an attractive novel therapeutic strategy for multiple maladies.

Autoantibodies, including rheumatoid factor (RF), are an important characteristic of rheumatoid arthritis (RA). Interestingly, several studies reported a correlation between the presence of IgA autoantibodies and worse disease course. We demonstrated previously that triggering the IgA Fc receptor (FcαRI) on neutrophils results in neutrophil recruitment and the release of neutrophil extracellular traps (NETs). Because this can lead to tissue damage, we investigated whether IgA immune complexes in plasma and synovial fluid of RA patients activate neutrophils. RF isotypes were measured with ELISA, and immune complexes were precipitated using polyethylene glycol 6000. Isolated neutrophils were incubated with immune complexes, and activation and release of NETs were determined in the presence or absence of FcαRI-blocking Abs. Plasma and SF of RA patients contained IgM, IgG, and IgA RFs. Patient plasma IgA RF and IgM RF showed a strong correlation. No uptake of IgM and minimal endocytosis of IgG immune complexes by neutrophils was observed, in contrast to avid uptake of IgA complexes. Incubation of neutrophils with immune complexes resulted in the production of reactive oxygen species, as well as the release of NETs, lactoferrin, and chemotactic stimuli. Importantly, activation of neutrophils was reduced when FcαRI was blocked. Neutrophils were activated by IgA immune complexes, which suggests that neutrophils play a role in inducing joint damage in RA patients who have IgA autoantibody complexes, thereby increasing the severity of disease. Blocking FcαRI inhibited neutrophil activation and, as such, may represent an additional attractive novel therapeutic strategy for the treatment of RA.

Many DNA vaccine candidates have been developed for the treatment of human papillomavirus type 16 (HPV16)-induced malignancies. Most of these vaccines consist of a fusion of E7 with a "carrier-protein" that functions to increase the potency of the vaccine. The nature of these carrier-proteins varies widely, and the mechanisms proposed to explain the enhanced immunogenicity of such fusions are often linked to the biological function of the carrier-protein. However, the potentiating effect of these carrier-proteins might also be explained by more general mechanisms, such as the provision of CD4+ T-cell help, increased antigen stability, or altered subcellular localization of the antigen. To assess whether these more generic mechanisms could suffice to generate highly immunogenic DNA vaccines, we evaluated a series of modular HPV16 E7 DNA vaccines in which the presence of CD4+ T-cell help, the presence of an endogenous carrier-protein, and the subcellular localization of the antigen could be systematically altered. Using this approach, we demonstrate that the addition of an element that provides CD4+ T-cell help, elements that enforce endoplasmic reticulum (ER) localization/retention are both necessary and sufficient to create markedly effective HPV16 E7-directed DNA vaccines. Importantly, the resulting design rules also apply to an HPV16 E6-directed DNA vaccine. The developed "HELP(ER)" HPV DNA vaccines encode only very limited additional sequences besides the antigen, thereby reducing the risk of antigenic competition and/or autoimmunity.