Antibodies generated against conserved antigens expressed by bacteria and allergen-bearing fungi suppress airway disease

Abstract

There has been a sharp rise in allergic asthma and asthma-related deaths in the developed world, in contrast to many childhood illnesses that have been reduced or eliminated. The hygiene hypothesis proposes that excessively sanitary conditions early in life result in autoimmune and allergic phenomena because of a failure of the immune system to receive proper microbial stimulation during development. We demonstrate that Abs generated against conserved bacterial polysaccharides are reactive with and dampen the immune response against chitin and Aspergillus fumigatus. A reduction in Ag uptake, cell influx, cell activation, and cytokine production occurred in the presence of anti-polysaccharide Abs, resulting in a striking decrease in the severity of allergic airway disease in mice. Overall, our results suggest that Ag exposure--derived from environmental sources, self-antigens, or vaccination--during the neonatal period has dramatic effects on the adult Ab response and modifies the development of allergic airway disease.

Figure 1. Monoclonal antibodies generated against conserved bacterial polysaccharides bind chitin and A. fumigatus

(A) Purified chitin particles (<10μm) were incubated with anti-GlcNAc IgM (HGAC78, blue), IgG3 (HGAC39, blue), or isotype control (Isotype, red) antibodies and analyzed by flow cytometry. (B) Live resting or 18 hr germinated A. fumigatus conidia were incubated with an IgM isotype control (Isotype, orange), anti-sialyllacto-N-tetraose (SMB19-IgM, red), anti-GlcNAc (GAC39-IgG3,blue), or anti-α-1,3 glucan (1-21-IgM, green) antibodies and analyzed by flow cytometry. (C) A. fumigatus conidia bound poly-L-lysine coated glass slides were incubated for 0, 4, 8, and 11 hrs in RPMI1640 + 2% FCS. Columns 1 and 3 show a field under phase contrast with germinated conidia (arrow), while columns 2 and 4 shows staining with SMB19 (red), GAC39 (blue), and 1-21 (green).

Figure 2. Anti-GlcNAc antibody dampens chitin-induced cellular recruitment and activation in the lung

C57BL/6 mice, treated i.v. with 5μg IgM isotype control or anti-GlcNAc antibodies (i.v. and i.t. in B), were challenged i.t. with 100μg of chitin particles. BAL was collected 24 hours after chitin challenge and the total numbers of neutrophils (Ly6G+) and alveolar macrophages (determined by their autofluorescence) (gating scheme shown in A) were enumerated by flow cytometry (B). Cytokine expression was measured by a Bio-plex cytokine array (C). Cells collected from collagenase-treated lungs and total cell numbers, neutrophils, and CD4+ T cells were calculated (D). Total RNA was collected from lung tissue-derived cells and analyzed by RT-PCR for CCL17 and CCL22 expression (E). Values represent the mean ± SEM from 3 independent experiments with 3-5 mice per group. Data were analyzed by a two-tailed unpaired t test. * = p<0.05, ** = p<0.01, *** = p<0.001 See also Figure S1

Figure 3. Anti-GlcNAc antibody decreases the uptake of chitin particles in vitro and in vivo

(A) Representative gating scheme for determining chitin positive cells. (B) Bone marrow derived macrophages (BMDM) and (C) A549 epithelial cells were cultured with 100μg of Alexa 647-labeled chitin, in the presence or absence of an IgM isotype control or anti-GlcNAc antibody, and chitin uptake was determined by flow cytometry. C57BL/6 mice, treated i.t. with an IgM isotype control or anti-GlcNAc antibodies, were challenged i.t. with 100μg of Alexa 647-labeled chitin particles and BAL was collected at 0, 1, 2, and 3 hours. Chitin positive (D) alveolar macrophages and (E) dendritic cells were detected by flow cytometry. Data represent the mean ± SEM from 3 independent experiments with 6 wells or 3-5 mice per group. Data were analyzed by a two-tailed unpaired t test. * = p<0.05, ** = p<0.01, *** = p<0.001

Figure 4. Anti-A. fumigatus antibodies dampen the allergic airway disease induced by A. fumigatus

(A) C57BL/6 mice were subjected to 5×10⁵ live A. fumigatus conidia, starting at 8 weeks of age, administered i.t. twice a week for 8 weeks. After one week without challenge, allergic airway disease was elicited using the same dose of A. fumigatus conidia, in the presence or absence of an isotype control, anti-GlcNAc antibody, or a combination of anti-GlcNAC, -sialyllactose-N-tetraose, -α-1,3 glucan), and lungs were analyzed 3 days later. (B) BAL and (C) cellular lung digests were collected and analyzed for neutrophil and eosinophil cell infiltration using flow cytometry. The level of (D) CD44 expression by CD4⁺ T cells and (E) total CD8⁺ T cells was measured by flow cytometry. Data represent the mean ± SEM from 3 independent experiments with 3-5 mice per group. Data were analyzed by a two-tailed unpaired t test. * = p<0.05, ** = p<0.01, *** = p<0.001 See also Figures S1.

Figure 5. Anti-A. fumigatus antibodies decrease the uptake of A. fumigatus in vitro and in vivo

(A) Representative gating scheme for determining A. fumigatus positive cells. (B) Bone marrow derived macrophages (BMDM) and (C) A549 epithelial cells were cultured with 1×10⁵ of Alexa 488-labeled A. fumigatus conidia, in the presence or absence of an IgM isotype control or an antibody combination (Ab combo) of anti-GlcNAC, -sialyllactose-N-tetraose, -α-1,3 glucan, and A. fumigatus uptake was measured by flow cytometry. C57BL/6 mice, treated i.t. with an IgM isotype control or antibody combo were challenged i.t. with 5×10⁵ of Alexa 488-labeled A. fumigatus conidia and BAL was collected at 0, 2, and 4 hours. (D) A. fumigatus-positive alveolar macrophages and (E) dendritic cells were determined using flow cytometry. Data represent the mean + SEM from 3 independent experiments with 6 wells or 3-5 mice per group. Data were analyzed by a two-tailed unpaired t test. * = p<0.05, ** = p<0.01, *** = p<0.001

Figure 6. Neonatal GAS immunization primes the adult antibody response and dampens the immune response to chitin

Neonatal mice were injected i.p. with PBS or GAS at 3 or 14 days of age. At 8 weeks of age, all groups were immunized i.v. with GAS and sera were collected before (preimmune) and 7 days (postimmune) after re-immunization. (A) The level of serum GlcNAc-specific IgM was determined by ELISA. (B) ELISPOT analysis was performed 7 days after the neonatal immunizations, Day 10 and Day 21 respectively, for GlcNAc-specific IgM ASCs. The post-immune mice were challenged i.t. with 100μg chitin (<10μm) 7 days following re-immunization at 8 weeks and the BAL level of GlcNAc-specific IgM (C) was determined by ELISA and total neutrophil influx (D) was determined by flow cytometry 24 hours following i.t. challenge. Data represent the mean + SEM from 3 independent experiments with 3-5 mice per group. Data were analyzed by a two-tailed unpaired t test. * = p<0.05, ** = p<0.01

Figure 7. Neonatal GAS immunization dampens the adult allergic airway disease induced by A. fumigatus

Neonatal C57BL/6 mice were immunized once i.p. with PBS or GAS at 3 days of age and grown up to 6-8 weeks before starting the chronic sensitization model outlined in Figure 4A. (A, B) BAL was collected 3 days following elicitation of the response and analyzed by flow cytometry for (A) neutrophils (neut.), eosinophils (eosin.), CD4⁺ T cells (CD4), total BAL cells and (B) mast cells and IgE+ B cells. Sera and BAL was analyzed for (C) total IgE, (D, E) GlcNAc-specific IgM and IgG3, and (F) A. fumigatus-specific total Ig against purified A. fumigatus allergic extract using ELISA. (G) Total lung leukocytes were stimulated with anti-CD3/CD28 in the presence of Brefeldin A and the percentage of CD4⁺IL-4⁺ cells was determined by flow cytometry. (H) Total RNA was collected from lung-derived cells and analyzed by RT-PCR for CCL11, 17, 22 and 24 expression. (I) BAL was analyzed for cytokine expression using the Bio-plex cytokine assay. Data were analyzed by an unpaired t test. * = p<0.05 See also Figures S1 and S2. Paraffin embedded lung sections from PBS and GAS immunized neonatal mice were stained with (J) hematoxylin and eosin (H&E) and (K) Alcian Blue-Periodic acid Schiff (AB-PAS). Data represent the mean ± SEM from 3 independent experiments with 3-5 mice per group.

Figure 8. Model for prevention from allergic airway disease by anti-PS antibodies

(A) This schematic shows our proposed model for the protection against A. fumigatus-induced allergic airway disease.