FcγR mediates TLR2- and Syk-dependent NLRP3 inflammasome activation by inactivated Francisella tularensis LVS immune complexes

Abstract

IgG (mAb)-opsonized, inactivated Francisella tularensis LVS (iFt-mAb) enhances TLR2-dependent IL-6 production by macrophages via Fcγ receptors (FcγR). In mice, vaccination with iFt-mAb provides IgA-dependent protection against lethal challenge with Ft LVS. Because inflammasome maturation of IL-1β is thought important for antibody-mediated immunity, we considered the possibility that iFt-mAb elicits an FcγR-dependent myeloid cell inflammasome response. Herein, we find that iFt-mAb enhances macrophage and dendritic cell IL-1β responses in a TLR2- and FcγR-dependent fashion. Although iFt-mAb complexes bind FcγR and are internalized, sensing of cytosolic DNA by absent in melanoma 2 (AIM2) is not required for the IL-1β response. In contrast, ASC, caspase-1, and NLR family pyrin domain-containing 3 (NLRP3) are indispensable. Further, FcγR-mediated spleen tyrosine kinase (Syk) signaling is required for this NLRP3-dependent IL-1β response, but the alternative IL-1β convertase caspase-8 is insufficient. Finally, iFt-mAb-vaccinated wild-type mice exhibit a significant delay in time to death, but IL-1R1- or Nlrp3-deficient mice vaccinated in this way are not protected and lack appreciable Francisella-specific antibodies. This study demonstrates that FcγR-mediated Syk activation leads to NLRP3 inflammasome-dependent IL-1β production in macrophages and suggests that an Nlrp3- and IL-1R-dependent process contributes to the IgA response important for protection against Ft LVS. These findings extend our understanding of cellular responses to inactivated pathogen-opsonized vaccine, establish FcγR-elicited Syk kinase-mediated NLRP3 inflammasome activation, and provide additional insight toward understanding crosstalk between TLR and FcγR signals.

Keywords: IL-1β; macrophage; vaccine.

Figure 1.. The iFt-mAb complex activates cytokine production in macrophages and BMDCs.

BMDM (A) and BMDC (B) from WT mice or PMA-treated THP-1 (C) were left untreated (media control) or stimulated for 24 h with iFt, mAb, or iFt-mAb at 5 µg/ml or at increasing concentrations. IL-1β and TNF-α were analyzed by ELISA using cell-free supernatants in duplicate. BMDM and THP-1 stimulations are means and sem of 3 independent experiments. BMDC results are means and sd of 2 independent experiments. Student’s t test was used to compare iFt to iFt-mAb. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.. The NLRP3 inflammasome is required for IL-1β production by iFt-mAb.

BMDM from WT, Casp-1 KO, or Asc KO (A), Aim2 KO (B), and Nlrp3 KO (C) mice were left untreated (media control) or stimulated with iFt or iFt-mAb or infected with Fn U112 (100 MOI) for 24 h before collection of cell-free culture supernatants for IL-1β and TNF-α ELISA. IL-1β and TNF-α analyzed by ELISA of THP-1– and NLRP3-deficient THP-1 24 h poststimulation with iFt or iFt-mAb. Control concentrations of IL-1β (pg/ml) for THP-1– and NLRP3-deficient THP-1 were 275.2 (sd 120.7) and 21.2 (sd 3.1), respectively (D). Results are means of 3 independent experiments with error bars indicating sem. *P < 0.05; ***P < 0.001.

Figure 3.. FcγR mediates inflammasome activation to enhance IL-1β.

(A) THP-1 were stimulated with iFt, mAb, or iFt-mAb for 23.5 h followed by treatment for 30 min with ATP (5 mM) before collection of cell-free culture supernatants for IL-1β or IL-18 by multiplex immunoassay. (B) Binding of GFP-iFt-mAb to THP-1 with or without FcγR blocking Abs as indicated by flow cytometry counting of cells labeled with GFP-iFt-mAb. The 1-dimensional histogram is 1 of 3 independent experiments with similar results. The filled curve is unstained cells, the solid curve is GFP-iFt-mAb without blocking Abs, the dashed curved is GFP-iFt-mAb with blocking Abs, and the dotted curve is GFP-iFt without mAb and without blocking Abs. THP-1 stimulated with iFt-mAb with and without FcγR blocking Abs were stained with Abs against mAbs (C). Green fluorescence indicates extracellular iFt-mAb; red fluorescence indicates intracellular iFt-mAb. Images were obtained by confocal microscopy. (D) Images are representative of 3 independent experiments with similar results. IL-1β and TNF-α from cell-free supernatants of THP-1 in the presence or absence of anti-FcγR blocking Abs. Shown are means of 3 independent experiments with error bars indicating sem. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4.. TLR2 and Src/Syk signaling are important for cytokine production with iFt-mAb.

IL-1β and TNF-α from cell-free supernatants of THP-1 stimulated for 24 h in the presence or absence of anti-TLR2 blocking Ab (A), with or without 30 min pretreatment with Syk (Pice, piceattenol, 5 µM) and Src inhibitors (PP1 and PP2, 10 μM) (B). Shown are means of 3 independent experiments with error bars indicating sem, except TNF-α in (B), which is the means of 2 independent experiments with triplicate samples and error bars indicating sd. *P < 0.05.

Figure 5.. Caspase-8 activity is necessary but insufficient for FcγR-mediated IL-1β.

BMDM from WT or Casp-1 KO mice were stimulated for 24 h with iFt or iFt-mAb, with or without pretreatment with a caspase-8 inhibitor (IETD), and IL-1β (A) and TNF-α (B) were analyzed by ELISA in cell-free supernatants. Caspase-8 activity in BMDM from WT or Casp-1 KO mice assayed using a luminescence detection system (C). Data shown reflect the means and sd of triplicate samples and are representative of 3 independent experiments with similar results. *P < 0.05; n.s., not significant.

Figure 6.. IL-1 and NLRP3 are important for survival against respiratory Ft-LVS in mice.

Groups of 10 WT, IL-1r1 KO, or Nlrp3 KO mice were immunized i.n. with either 20 μl of vehicle (PBS) or 20 μl of 2 × 10⁷ inactivated organisms complexed with 1 μg/ml mAb (iFt-mAb) in PBS (Vacc). Mice were immunized on d 0 and d 21 and then challenged i.n. on d 35 with 20,000 CFU live Ft-LVS and monitored for survival (A, C, D, E, F, and G). Survival curves in all 6 of these panels are from the same groups of mice and are separated for ease of comparison. Results from 1 of 2 vaccination studies with similar results are shown. Groups of 6 WT or IL-1R1 KO mice were infected i.n. with 500 CFU Ft LVS and observed for survival (B). Serum from WT and IL-1r1 mice was collected before challenge, and relative Ft LVS-specific Ab levels for IgA and IgG were measured by ELISA (H). Serum from WT and Nlrp3 mice was collected before challenge, and relative Ft LVS-specific Ab levels for IgA and IgG were measured by ELISA (I). The bars represent the average of Ft LVS-specific Ab detected in serum of iFt-mAb treated mice normalized to the corresponding average unvaccinated serum Ab level. Survival curves were compared using the log-rank test (Mantel-Cox). *P < 0.05; N.S., not significant.