IgG1 is pathogenic in Leishmania mexicana infection

Abstract

There are >2 million new cases of leishmaniasis annually, and no effective vaccine has been developed to prevent infection. In murine infection, Leishmania mexicana, which lives intracellularly in host macrophages, has developed pathways to hijack host IgG to induce a suppressive IL-10 response through FcγRs, the cell-surface receptors for IgG. To guide vaccine development away from detrimental Ab responses, which can accompany attempts to induce cell-mediated immunity, it is crucial to know which isotypes of IgG are pathogenic in this infection. We found that IgG1 and IgG2a/c induce IL-10 from macrophages in vitro equally well but through different FcγR subtypes: IgG1 through FcγRIII and IgG2a/c through FcγRI primarily, but also through FcγRIII. In sharp contrast, mice lacking IgG1 develop earlier and stronger IgG2a/c, IgG3, and IgM responses to L. mexicana infection and yet are more resistant to the infection. Thus, IgG1, but not IgG2a/c or IgG3, is pathogenic in vivo, in agreement with prior studies indicating that FcγRIII is required for chronic disease. This calls into question the assumption that macrophages, which should secrete IL-10 in response to IgG1 and IgG2a/c immune complexes, are the most important source of IL-10 generated by IgG-FcγR engagement in L. mexicana infection. Further investigations are required to better determine the cell type responsible for this immunosuppressive FcγRIII-induced IL-10 pathway and whether IgG2a/c is protective.

FIGURE 1. IgG1 and IgG2a/c can induce IL-10 from macrophages through FcγRIII, and FcγRI and III respectively, but FcγRIV is not involved

BMMΦs were prepared from B6 WT, FcγRIII KO, FcγRI KO, and FcRγ KO mice (strains are numbered 1-4) and incubated with anti-IL-10R and with the following additions (conditions are labeled A-J): media; LPS; chicken OVA-rabbit anti-OVA complexes (IgG-OVA); unopsonized AAs; or AAs opsonized with uninfected serum (uninf-AA) or anti-L. mexicana IgG1 serum (IgG1 serum-AA), IgG2a/c serum (IgG2a/c serum-AA), purified IgG1 (pure IgG1-AA), or IgG2a/c mAb 3E4-G1-G4 (IgG2a/c mAb-AA) for 20 h, and IL-10 was measured in the supernatants by ELISA. IgG-OVA + LPS was used as a positive control. Opsonized parasites generate similar amounts of IL-10 to unopsonized parasites when LPS is not present. IgG-OVA also does not induce significant amounts of IL-10 when LPS is absent. A, IgG1-induce IL-10 with controls. p < 0.05 for 1B versus 1E, 1F; 1F versus 1G, 1H; 2F versus 2G; 4G versus 1G; 4H versus 1H; 2G versus 1G; 3G versus 2G; 4G versus 2G; 2H versus 1H; and 3H versus 2H. B, IgG2a/c-induced IL-10 with controls. p < 0.05 for 1B versus 1E, 1F; 1F versus 1I, 1J; 3F versus 3I; 4I versus 1I; 4J versus 1J; 3I versus 1I; 4I versus 1I; 3J versus 1J; and 4J versus 1J. C, BMMΦs were prepared from B6 WT and FcγRIII KO mice and incubated with anti-IL-10R and media alone, LPS, unopsonized AAs, AAs with LPS (AA + LPS), or AAs opsonized with IgG2a/c serum with LPS (IgG2a/c-AA + LPS) for 20 h, and IL-10 was measured in the supernatants by ELISA. Anti-FcγRIV-blocking Ab (9E9; 20 μg/ml) was added to the cultures indicated. No statistically significant differences were seen between B6 and FcγRIII KO cells or for IgG2a/c-AA + LPS in the presence or absence of anti-FcγRIV-blocking Ab.

FIGURE 2. IgG1 KO mice are more resistant to L. mexicana infection with an earlier IFN-γ response than WT mice

A, IgG1 KO and B6 WT mice were infected in the right hind footpad with 5×10⁶ stationary-phase L. mexicana promastigotes, and lesion size was monitored. Lesion sizes were different at 15 wk and thereafter (p < 0.05). B, At the times indicated post-infection, lesion parasite burdens from IgG1 KO and B6 WT mice were determined by limiting dilution. C, At the times indicated post-infection, draining LN cells were stimulated with FTAg for 3 d, and supernatants were assayed for IFN-γ. *p < 0.05.

FIGURE 3. IgG1, IgG2a/c, and IgM levels in IgG1 KO and B6 mice infected with L. mexicana

IgG1 KO and B6 WT mice were infected as in Fig. 2, and serum samples collected at the times shown were evaluated for L. mexicana-specific IgM (A), IgG1 (B), and IgG2a/c (C) by ELISA. In C, the ratio of relative abundances (IgG1 KO/B6 WT) is shown for each time point. *p < 0.05 (A-C). D, Sera from IgG1 KO and B6 WT mice infected with L. mexicana for 12 wk were depleted of IgG1 and then ELISAs were performed for IgG2a/c. *p < 0.05 for IgG1 KO versus other groups; p > 0.05 for B6 WT versus B6 WT-IgG1 depleted (paired t test). Undepleted samples received streptavidin beads without biotinylated anti-IgG1.

FIGURE 4. IgG1 KO mice have stronger and earlier IgG3 responses

A, IgG1 KO and B6 WT mice were infected, as in Fig. 2 and serum samples (collected at the times shown) were evaluated for L. mexicana-specific IgG3 by ELISA. *p < 0.05 at all dilutions. B, The kinetics of L. mexicana-specific IgG3 levels were determined. The ratio of relative abundances (IgG1 KO/B6 WT) is shown for each time point. *p < 0.02. C, Sera from IgG1 KO and B6 WT mice infected with L. mexicana for 12 wk were depleted of IgG1 and then ELISAs were performed for IgG3. *p < 0.05 for IgG1 KO versus other groups; p > 0.05 for B6 WT versus B6 WT-IgG1 depleted (paired t test). Undepleted samples received streptavidin beads without biotinylated anti-IgG1.