IgE Effector Mechanisms, in Concert with Mast Cells, Contribute to Acquired Host Defense against Staphylococcusaureus

Abstract

Allergies are considered to represent mal-directed type 2 immune responses against mostly innocuous exogenous compounds. Immunoglobulin E (IgE) antibodies are a characteristic feature of allergies and mediate hypersensitivity against allergens through activation of effector cells, particularly mast cells (MCs). Although the physiological functions of this dangerous branch of immunity have remained enigmatic, recent evidence shows that allergic immune reactions can help to protect against the toxicity of venoms. Because bacteria are a potent alternative source of toxins, we assessed the possible role of allergy-like type 2 immunity in antibacterial host defense. We discovered that the adaptive immune response against Staphylococcus aureus (SA) skin infection substantially improved systemic host defense against secondary SA infections in mice. Moreover, this acquired protection depended on IgE effector mechanisms and MCs. Importantly, our results reveal a previously unknown physiological function of allergic immune responses, IgE antibodies, and MCs in host defense against a pathogenic bacterium.

Keywords: IgE; Staphylococcus aureus; allergy; allergy module; bacteria; basophils; degranulation; host defense; mast cells; type 2 immunity.

Figure 1:

Skin-initiated immunity increases host defense against severe soft tissue infection with SA (A) Experimental scheme for B to G. (B) Day 21 serum amounts of SA culture supernatant (SA-SN)-specific IgG1 (SA-SN IgG1) and total IgE antibodies. (C) Day 21 serum amounts of SA-SN-specific IgE (SA-SN IgE). (D) Degranulation (% ß-hexosaminidase release) upon SA-SN exposure of fetal skin-derived cultured mast cells (FSCMCs) sensitized with untreated or anti-IgE-treated serum from PBS or SA-infected animals. (E and F) Bacterial colony forming units (CFUs) in (D) ear and (E) draining cervical lymph nodes 20 hours after ear skin and soft tissue (ESST) infection. (G) Mice with ear tissue loss on day 6 after ESST infection and representative (of two independent experiments) pictures. (H) Experimental scheme for (I). (I) Day 21 (primary immune response) and day 42 (secondary immune response) serum amounts of SA-SN-specific IgG1, IgG2b, IgG2c (SA-SN IgG1 IgG2b, IgG2c, respectively) and total IgE antibodies. (B to F, I): Mann-Whitney Test for indicated pairwise comparisons; symbols represent biological replicates; (B): n=9-10 (only mock-infected samples with an IgG1 titer ≥ 1 are shown); (C): n=10-12; (E and F): n=12-16; (G): n=5; (I): n=12 (only mock-infected samples with an IgG1 titer ≥ 1 are shown); data shown were pooled from two independent experiments, except for (D), which are from one experiment (representative of two independent experiments). ns (not significant); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Error bars represent mean +SEM apart from (D) where they represent mean +SD. Please also see Figure S1.

Figure 2:

Prior skin infection improves host defense against SA pneumonia (A) Experimental scheme for B to G. (B) Lung tissue cytokine amounts 6 hours after infection. (C) Lung immune cell counts (by flow cytometry) 6 hours after infection (also see Figure S2 for gating strategy and additional cell types). (D) lung (homogenate) cytokine amounts (E) body temperature, and (F) lung tissue CFUs 18 hours after infection. (G) Survival after intranasal infection. (C to G): Mann-Whitney Test for indicated pairwise comparisons; symbols represent biological replicates; (G): Mantel-Cox test; (G): n=13-14; (B to E): n=11-14; (B): n=7; (C): n=15; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Error bars represent mean + or −SEM apart from (F) where they represent mean +SD. Please also see Figure S2.

Figure 3:

Acquired immunity against SA requires functional IgE effector mechanisms (A) Experimental scheme for B to I. (B to G) Bacterial colony forming units (CFUs) in (B, D and F) ears and (C, E and G) draining (cervical) lymph nodes 20 hours after ear skin and soft tissue (ESST) infection. (H) Mice with ear tissue loss on day 6 after ESST infection. (I) Quantification of none (−), moderately (+) and extensively (++) degranulated mast cells in whole ear tissue sections 6 hours after ESST infection. (B to G, I): Mann-Whitney Test for indicated pairwise comparisons; symbols represent values from single mice; (B): data were pooled from two independent experiments. (I): data are from one experiment (representative of two independent experiments). (B and C) n=10-16; (D and E) n=17-18; (F and G) n=11-13; ns (not significant); (H) n=5-6; (I) n=4-6; ns (not significant); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Error bars represent (B to G) mean +SEM or (I) mean +SD. Please also see Figure S3.

Figure 4:

IgE effector mechanisms contribute to acquired immunity against SA pneumonia (A) Experimental scheme for B to E. (B and D) Body temperature and (C and E) lung tissue bacterial colony forming units (CFUs) 18 hours after intranasal infection. (B to E): Mann-Whitney Test for indicated pairwise comparisons; symbols represent values from single mice; (D and E): data were pooled from two independent experiments. (D and E) n=11-14; (B and C) data shown are from one experiment; n=4-8. ns (not significant); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Error bars represent (D and E) mean + or − SEM or (B and C) mean + or − SD.

Figure 5:

MCs and local allergic reactions can contribute to anti-SA immunity (A) Experimental scheme for B to E. (B to E) Bacterial colony forming units (CFUs) in the (B and D) ears and (C and E) draining (cervical) lymph nodes 20 hours after ear skin and soft tissue (ESST) infection. (F) Mice with ear tissue loss on day 6 after ESST infection. (G) Experimental scheme for H and I. (H) Body temperature and (I) lung tissue bacterial CFUs 18 hours after intranasal infection. (J) Experimental scheme for K to M. (K) Ear swelling 30 minutes after dinitrophenyl-labelled human serum albumin (DNP-HSA) injection in mice that received anti-DNP IgE the day before. (L and M) Bacterial load in (L) ear and (M) draining lymph nodes 20 hours after ESST infection and DNP-HSA injection. (B to E, H, I, K-M): Mann-Whitney Test for indicated pairwise comparisons; all graphs show data pooled from two independent experiments apart from H and I which depict results of one experiment; symbols represent values from single mice; (B and C) n=14-15; (D and E) n=10-12; (F) n=5-7; (H and I) n=5-7; (K) n=10-11; (L and M) n=16-18. ns (not significant); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Error bars represent mean +SEM. Please also see Figure S4.

Figure 6:

IgE-activated MCs counteract SA toxicity and amplification (A) Cytokine production of fetal skin-derived cultured mast cells (FSCMCs) sensitized with serum from mice with prior epicutaneous SA or mock infection upon exposure to SA culture supernatant (SA-SN) for 18 hours. (B) SA viability after 6 hours co-culture with or without FSCMCs pre-sensitized or not with immune serum collected from mice three weeks after epicutaneous back skin infection. (C) Viability (% of untreated cells) of A549 lung epithelial cells after 18 hours exposure to SA-SN (left panel) or plain bacterial culture medium (right panel) which was either untreated or pre-incubated with supernatant collected from anti-dinitrophenyl (DNP) IgE-sensitized or non-sensitized FSCMCs 1 h or 20 h after DNP-labelled human serum albumin (DNP-HSA) stimulation. (D) SA viability after 6 hours culture in presence of supernatant of FSCMCs that were stimulated (or not) with IgE and antigen, containing mediators released over one (0-1 h), six (0-6 h) or 20 hours (0-20 h) (left panel). Right panel: bacterial viability upon culture with supernatant from mast cells from which supernatant was removed after 1 hour (1-20 h) or 6 hours (6-20 h), followed by addition of fresh medium and antigen for the remaining time. (E) Volcano plot depicting mass spectrometry results of SN collected 1 h after DNP-HSA exposure of anti-DNP IgE-sensitized or non-sensitized FSCMCs. Significantly up-regulated data points (blue circles) with potential antibacterial function are annotated. (F) Enriched GO term molecular functions among up-regulated proteins (see E). (G) SA viability 6 hours after culture with SN from non-stimulated or IgE- and antigen- (1 hour) stimulated FSCMCs. Supernatant of stimulated cells was optionally treated with protease inhibitor. (A to D, G): Mann-Whitney Test for indicated pairwise comparisons; (A to D, G): Data are representative of at least two independent experiments. (E and F) Data are representative of one analysis of sample triplicates analyzed in duplicate. ns (not significant); *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (A to D, G) Error bars represent mean +SD. Please also see Figure S5.