Chronic Infection by Mucoid Pseudomonas aeruginosa Associated with Dysregulation in T-Cell Immunity to Outer Membrane Porin F

Abstract

Rationale: Pseudomonas aeruginosa (PA) is an environmental pathogen that commonly infects individuals with cystic fibrosis (CF) and non-CF bronchiectasis, impacting morbidity and mortality. To understand the pathobiology of interactions between the bacterium and host adaptive immunity and to inform rational vaccine design, it is important to understand the adaptive immune correlates of disease.

Objectives: To characterize T-cell immunity to the PA antigen outer membrane porin F (OprF) by analyzing immunodominant epitopes in relation to infection status.

Methods: Patients with non-CF bronchiectasis were stratified by frequency of PA isolation. T-cell IFN-γ immunity to OprF and its immunodominant epitopes was characterized. Patterns of human leukocyte antigen (HLA) restriction of immunodominant epitopes were defined using HLA class II transgenic mice. Immunity was characterized with respect to cytokine and chemokine secretion, antibody response, and T-cell activation transcripts.

Measurements and main results: Patients were stratified according to whether PA was never, sometimes (<50%), or frequently (≥50%) isolated from sputum. Patients with frequent PA sputum-positive isolates were more likely to be infected by mucoid PA, and they showed a narrow T-cell epitope response and a relative reduction in Th1 polarizing transcription factors but enhanced immunity with respect to antibody production, innate cytokines, and chemokines.

Conclusions: We have defined the immunodominant, HLA-restricted T-cell epitopes of OprF. Our observation that chronic infection is associated with a response of narrowed specificity, despite strong innate and antibody immunity, may help to explain susceptibility in these individuals and pave the way for better vaccine design to achieve protective immunity.

Keywords: Pseudomonas; T lymphocyte; adaptive immunity; bronchiectasis; epitopes.

Figure 1.

T-cell and serum antibody responses to Pseudomonas aeruginosa (PA) protein outer membrane porin F (OprF) in patients with non–cystic fibrosis bronchiectasis. (A) The magnitude of the T-cell response to OprF and control anti-CD3 in the three bronchiectasis groups classified according to the PA infection status were determined (never [0%], n = 31; sometimes [<50%], n = 12; and frequent [≥50%], n = 14 culture positive for PA). In the group that were less than 50% sputum sample culture positive for PA, 4 of 12 (25%) individuals grew mucoid isolates, and in the greater than or equal to 50% group, 11 of 14 (79%) individuals grew mucoid isolates (chi-square test, 5.42; P = 0.0199, Pearson). (B) The magnitude of the T-cell response to OprF and control anti-CD3 was measured and plotted according to the PA isolate culture status (never [0%], n = 31; nonmucoid, n = 11; or mucoid, n = 15). (C) The IgG OprF-specific antibody titers in the three bronchiectasis groups classified according to the PA infection status were determined (never [0%], n = 45; sometimes [<50%], n = 16; and frequent [≥50%], n = 21). (D) The IgG OprF-specific antibody titer was measured and plotted according to the PA isolate culture status (never [0%], n = 45; nonmucoid, n = 17; or mucoid, n = 19). Data are presented ±SEM. Statistical significance was determined using the Kruskal-Wallis test followed by a post hoc Dunn test. **P < 0.01, ***P < 0.001. PBMC = peripheral blood mononuclear cells; sfu = spot-forming unit.

Figure 2.

T-cell epitope responses to outer membrane porin F (OprF) peptide panel in patients with non–cystic fibrosis bronchiectasis with evidence of chronic Pseudomonas aeruginosa (PA) infection. Epitope mapping of patient T-cell responses to OprF were determined by screening responses to an overlapping peptide panel of OprF for (A) PA infection status (never [0%], n = 31; sometimes [<50%], n = 12; and frequent [≥50%], n = 14 culture positive) or (B) PA isolate (never PA positive, n = 31; nonmucoid, n = 11; or mucoid, n = 15). Representative examples of patient T-cell responses to the peptide panel are shown for the never (0%) (C), sometimes (<50%) (D), and frequently (≥50%) (E) culture positive. Two SD above the mean of the media-only control is shown as a horizontal dotted line. (F) The overlapping relationships of epitopes identified in bronchiectasis patients with PA isolates classified as never PA culture positive (n = 31) and nonmucoid (n = 11) and mucoid (n = 15) positive cultures visualized as a Venn diagram. Data are presented ±SEM. Statistical significance was determined using the Kruskal-Wallis test followed by a post hoc Dunn test. *P <  0.05, **P < 0.01. PBMC = peripheral blood mononuclear cell; sfc = spot-forming cell.

Figure 3.

CD4 T-cell epitope responses to outer membrane porin F (OprF) peptide panel in HLA class II transgenic mice. Draining lymph nodes from transgenic mice for DRB1*0101, n = 15 (A), DRB1*0401, n = 9 (B), and DRB1*1501, n = 10 (C) were harvested 10 days postimmunization with OprF protein in complete Freund’s adjuvant. Recall peptide responses were determined using the overlapping peptide panel for OprF. Responses were considered positive if the response was greater than 2 SD above the mean of the negative control (see horizontal dotted line). SEB = staphylococcal enterotoxin B; SFC = spot-forming cell.

Figure 4.

Cytokine and chemokine responses to outer membrane porin F protein in non–cystic fibrosis bronchiectasis with evidence of chronic Pseudomonas aeruginosa infection. Proinflammatory cytokine concentrations for (A) IL-6, (B) macrophage inflammatory protein (MIP)-1α, (C) MIP-1β, (D) IL-12, and (E) IL-8 were determined from the supernatant of peripheral blood mononuclear cells stimulated with outer membrane porin F. Concentrations were measured using the 30-plex Luminex assay in patients with bronchiectasis classified as never (0%; n = 13 for IL-6, MIP-1α, and IL-8; n = 24 for MIP-1β and IL-12) or frequently (≥50%; n = 12 for IL-6, MIP-1α, and IL-8; n = 8 for MIP-1β and IL-12) culture positive for P. aeruginosa. Data are presented ±SEM. Statistical significance was determined using the Mann-Whitney U test. *P < 0.05, **P < 0.01.

Figure 5.

Lymphocyte transcriptional changes in patients with non–cystic fibrosis bronchiectasis with evidence of chronic Pseudomonas aeruginosa infection. The expression of (A) T-box transcription factor 21 (TBX21), (B) S1P receptor 1 (S1pr1), (C) retinoic acid receptor–related orphan receptor cT (RORcT), (D) trans-acting T cell–specific transcription factor (Gata3), (E) forkhead box P3 (FoxP3), and (F) IL-10 were determined by quantitative polymerase chain reaction. Peripheral blood mononuclear cells were stimulated with phorbol myristate acetate/ionomycin, from which RNA was isolated and converted to cDNA. Patients with broncheiectasis were classified as never (0%; n = 7) or frequently (>50%; n = 4) culture positive for P. aeruginosa. Data are presented ±SEM. Statistical significance was determined using the Mann-Whitney U test. *P < 0.05.