Quaternary Ammonium Compound Disinfectants Reduce Lupus-Associated Splenomegaly by Targeting Neutrophil Migration and T-Cell Fate

Abstract

Hypersensitivity reactions and immune dysregulation have been reported with the use of quaternary ammonium compound disinfectants (QACs). We hypothesized that QAC exposure would exacerbate autoimmunity associated with systemic lupus erythematosus (lupus). Surprisingly, however, we found that compared to QAC-free mice, ambient exposure of lupus-prone mice to QACs led to smaller spleens with no change in circulating autoantibodies or the severity of glomerulonephritis. This suggests that QACs may have immunosuppressive effects on lupus. Using a microfluidic device, we showed that ambient exposure to QACs reduced directional migration of bone marrow-derived neutrophils toward an inflammatory chemoattractant ex vivo. Consistent with this, we found decreased infiltration of neutrophils into the spleen. While bone marrow-derived neutrophils appeared to exhibit a pro-inflammatory profile, upregulated expression of PD-L1 was observed on neutrophils that infiltrated the spleen, which in turn interacted with PD-1 on T cells and modulated their fate. Specifically, QAC exposure hindered activation of splenic T cells and increased apoptosis of effector T-cell populations. Collectively, these results suggest that ambient QAC exposure decreases lupus-associated splenomegaly likely through neutrophil-mediated toning of T-cell activation and/or apoptosis. However, our findings also indicate that even ambient exposure could alter immune cell phenotypes, functions, and their fate. Further investigations on how QACs affect immunity under steady-state conditions are warranted.

Keywords: T cells; autoimmunity; lupus (SLE); neutrophil; quaternary ammonium compound (QAC); splenomegaly.

Figure 1

Reduced lupus-associated splenomegaly with ambient exposure to QACs. (A) Study design of the 1st experimental setting. See text for details. (B) Weight of lymphoid organs shown as ratios to the body weight at 16 weeks of age. RLN, renal lymph node. MLN, mesenteric lymph node. (C) Study design of the 2nd experimental setting. (D) Weight of lymphoid organs shown as ratios to the body weight at 12 weeks of age. Data are represented as mean ± SEM. ^≠P < 0.10, *P < 0.05.

Figure 2

Decreased ex-vivo migration and chemotaxis of neutrophils with ambient exposure to QACs. (A) Pattern of the competitive chemotaxis toward LTB₄ as determined by a microfluidic μC3 assay. (B) Percentage of migrated neutrophils toward LTB₄. (C) Representative micrographs showing directional (straight channels) vs. non-directional migration (cells were lost within the maze). Data obtained from 12-week-old mice are shown as mean ± SEM. n = 5/group, *P < 0.05.

Figure 3

Decreased infiltration of neutrophils into the spleen with ambient exposure to QACs. (A) Splenic Gr-1+ cells as the percentage of CD11b+CD11c− cells (left) and Gr-1+ myeloid cells as the percentage of total leukocytes (right). (B) Representative micrographs of IHC-stained splenic sections showing Ly6G+ neutrophils. (C) Quantification of the micrographs shown as the mean intensity of PE fluorescence. (D) Serum level of TNFα. (E) Transcript level of CXCR4 in BM-enriched neutrophils. (F) Ratio of the transcript levels of CXCR4 and CXCR2. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Pro-inflammatory neutrophilic phenotype with ambient exposure to QACs. Neutrophils were enriched from the bone marrow and relative transcript levels of (A) IL-6, (B) IL-1β, (C) TNFα, and (D) BAFF are shown. Data from the 1^st experimental setting are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Reduced expansion and activation of splenic T cells with ambient exposure to QACs. (A) Percentage of splenic Gr-1+ myeloid cells expressing PD-L1. (B) Neutrophilic transcript level of PD-L1. (C–D) Quantification of the mean intensity of PD-L1 (C) and PD-1 (D) fluorescence in IHC-stained splenic sections. (E) Representative micrographs of IHC-stained splenic sections showing the co-localization of T-cell (CD3) expression of PD-1 and neutrophil (Ly6G) expression of PD-L1. (F) Percentage of CD3+ splenic T cells in total live cells. (G) Percentage of CD8+ cells in splenic T cells. (H) The ratio of CD4/CD8. (I) Percentage of CD4+ T cells expressing the activation marker CD69. Data are represented as mean ± SEM. ^≠P < 0.10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

Increased apoptosis of T cells with ambient exposure to QACs. (A) Percentage of early apoptotic T_CM cells gated as AnnexinV+PI− on CD3+CD62L+CD44+ cells. (B) Percentage of late apoptotic or necrotic T_EM cells gated as AnnexinV+PI+ on CD3+CD62L-CD44+ cells. (C) Ratio of early apoptotic (AnnexinV+PI−) to late apoptotic or necrotic (AnnexinV+PI+) T_EM cells. (D) Percentage of early and late apoptotic DN-T cell as the percentage of CD3+ T lymphocytes. (E) Serum level of IL-6. (F) Transcript level of IL-6 in whole spleen. Data from the 1st experimental setting are shown as mean ± SEM. ^≠P < 0.10, *P < 0.05, **P < 0.01.