Immunogenic properties of archaeal species found in bioaerosols

Abstract

The etiology of bioaerosol-related pulmonary diseases remains poorly understood. Recently, archaea emerged as prominent airborne components of agricultural environments, but the consequences of airway exposure to archaea remain unknown. Since subcomponents of archaea can be immunogenic, we used a murine model to study the pulmonary immune responses to two archaeal species found in agricultural facilities: Methanobrevibacter smithii (MBS) and Methanosphaera stadtmanae (MSS). Mice were administered intranasally with 6.25, 25 or 100 µg of MBS or MSS, once daily, 3 days a week, for 3 weeks. MSS induced more severe histopathological alterations than MBS with perivascular accumulation of granulocytes, pronounced thickening of the alveolar septa, alveolar macrophages accumulation and increased perivascular mononucleated cell accumulation. Analyses of bronchoalveolar lavage fluids revealed up to 3 times greater leukocyte accumulation with MSS compared to MBS. Instillation of 100 µg of MBS or MSS caused predominant accumulation of monocyte/macrophages (4.5×10(5) and 4.8×10(5) cells/ml respectively) followed by CD4(+) T cells (1.38×10(5) and 1.94×10(5) cells/ml respectively), B cells (0.73×10(5) and 1.28×10(5) cells/ml respectively), and CD8(+) T cells (0.20×10(5) and 0.31×10(5) cells/ml respectively) in the airways. Both archaeal species induced similar titers of antigen-specific IgGs in plasma. MSS but not MBS caused an accumulation of eosinophils and neutrophils in the lungs, which surprisingly, correlated inversely with the size of the inoculum. Stronger immunogenicity of MSS was confirmed by a 3 fold higher accumulation of myeloid dendritic cells in the airways, compared to MBS. Thus, the dose and species of archaea determine the magnitude and nature of the pulmonary immune response. This is the first report of an immunomodulatory role of archaeal species found in bioaerosols.

Figure 1. Experimental model.

Mice received intranasal instillations of saline or; 6.25 µg, 25 µg, 100 µg of MBS or; 6.25 µg, 25 µg, 100 µg of MSS, once daily, starting on day 0, for 3 consecutive days in a week, during 3 weeks. Mice were euthanized on day 20, 4 days after last archaea instillation.

Figure 2. MBS and MSS induce dose-dependent histopathological alterations in the lungs.

Hematoxylin and eosin-stained sections of the lungs obtained from A) mice instilled with saline 3 times a week for 3 weeks; mice instilled with B) 6.25, C) 25 or D) 100 µg of MBS 3 times a week for 3 weeks; mice instilled with E) 6.25, F) 25 or G) 100 µg of MSS 3 times a week for 3 weeks. MSS strongly induces the formation of tertiary lymphoid organ-like structures (arrows) compared to MBS. Specimens shown are representative of 6 individual observations per group.

Figure 3. Quantification of leukocyte subsets in the BALF of mice instilled with increasing doses of two archaeal species.

A) Total immune cell numbers in BALF of mice instilled with 6.25, 25 or 100 µg of MBS or MSS for 3 weeks. B) Leukocyte subset numbers in BALF of mice exposed to 6.25, 25 and 100 µg of MBS for 3 weeks. C) Leukocyte subset numbers in BALF of mice exposed to 6.25, 25 and 100 µg of MSS for 3 weeks. Results expressed as average ± SEM. For both archaeal species, BALF cell subtypes were mainly composed of macrophages and lymphocytes. MSS also induced a significant accumulation of eosinophils and of neutrophils. Letters a and b represent statistical differences between treatment regimens within a same cell type. Results from two pooled experiments obtained with similar results are presented and 14 to 22 mice per group were analyzed. p<0.05.

Figure 4. Dominance of CD4⁺ T lymphocytes and CD19⁺ B lymphocytes in BALF of mice instilled with archaeal species.

The numbers of CD4⁺ T cells, CD8⁺ T cells and CD19⁺ B cells were analyzed in BALF of mice exposed for 3 weeks to 6.25, 25 and 100 µg of A) MBS or B) MSS. Compared to MBS, MSS induced strong CD4⁺ helper T cell and CD19⁺ B cell responses in the airways, which plateaued from the lowest dose instilled. Results are expressed as median ± interquartile intervals. Letters a and b represent statistical differences between treatment regimens within a same cell type. Six mice per group were analyzed. p<0.05.

Figure 5. MBS and MSS induce a dose-dependent production of antigen-specific IgG in plasma.

Titers of antigen-specific IgG were measured by ELISA in plasma of mice exposed to 6.25, 25 and 100 µg of A) MBS or B) MSS. C) Titers are expressed as the logarithmic inverse of plasma dilutions. Both archaea induced a dose-responsive generation of archaea-specific antibodies. Results are expressed as mean ± SEM. Letters a and b represent statistical differences between treatment regimens. Six mice per group were analyzed. p<0.05.

Figure 6. MSS induces stronger myeloid dendritic cells response than MBS in the airways of mice.

Flow cytometric analyses were performed on BALF cells of mice exposed to 6.25 or 100 µg of MBS or MSS for 3 weeks. Compared to saline-treated mice, MBS did not induce significant accumulation of myeloid dendritic cells in the airways. Myeloid dendritic cell response was increased with MSS. Results are expressed as absolute numbers of cells (average ± SEM). Six mice per group were analyzed. a: p<0.05, represent statistically differences compared to saline. b: p = 0.08.