Virginia Research Day 2021
Quaternary ammonium compound disinfectants reduce lupus-associated splenomegaly by targeting neutrophil migration and t-cell fate Leila Abdelhmaid 1 , Xavier Cabana-Puig 1 , Qinghui Mu 1,2 , Maryam Miarefian 3 , Brianna Swartwout 4 , Kristin Eden 1 , Prerna Das 1 , Ryan P. Seguin 5 , Libin Xu 5 , Sarah Lowen 6 , Mital Lavani 6 , Terry C. Hrubec 1,6* , Caroline N. Jones 7,8* and Xin M. Luo 1* 1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 2 School of Medicine, Stanford University, 3 Department of Mechanical Engineering, VT, 4 TBMH Graduate Program, VT, 5 Department of Medicinal Chemistry, School of Pharmacy, University of Washington, 6 Department of Anatomical Sciences, Edward Via College of Osteopathic Medicine, 7 Department of Biological Sciences, VT, 8 Department of Bioengineering, University of Texas.
ABSTRACT
RESULTS
RESULTS
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 (SLE or 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. Research Questions, Objectives & Study design
Reduced Expansion and Activation of Splenic T Cells
With Ambient Exposure to QACs:
(A)
* Reduced Lupus-Associated Splenomegaly (B)
(B)
(C)
(A)
Figure 3. Reduced lupus-associated splenomegaly with ambient exposure to QACs. (A) Weight of spleens shown as ratios to the body weight at 16 weeks of age, of animal of the 1 st experimental setting. (B) Weight of spleens shown as ratios to the body weight at 12 weeks of age, of animal of the 2 nd experimental setting.
0.020
**
***
*
5
10 15 20 25 30 35 40 45
2.5
4
0.015
1 %Of CD3 + 2 3
2.0
1.5
0.010
(g)
1.0
(g)
0 5
0
0.005
CD8 +
CD3 + % Of total live cells
0.5
Spleen/BWT
(D)
(E)
Spleen/BWT
0.000
0.0
*
**
50
0 10 20 30 40 50 60 70 80
Decreased Ex Vivo Migration and Chemotaxis of Neutrophils
40
10 CD4 + /CD8 + 20 30
% Of CD4 +
(C)
(A)
(B)
QAC-Free
0 4 %Neutrophil Migration toward LTB 4 * 1 2 3
12
0
10
CD69 +
8
100µm
Figure 7. Reduced expansion and activation of splenic T cells with ambient exposure to QACs. (A) 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. (B) Percentage of CD3+ splenic T cells in total live cells. (C) Percentage of CD8+ cells in splenic T cells. (D) The ratio of CD4/CD8. (E) Percentage of CD4+T cells expressing the activation marker CD69.
6
QAC-Exposed
4
2
0 50 100 150 200 250 300 350 400 0 Time (min) %Neutrophil Migration toward LTB4
Increased Apoptosis of T cells
100µm
(A)
(B)
(C)
Figure 4. Decreased ex-vivo migration and chemotaxis of neutrophils with ambient exposure to QACs. (A) Pattern of the competitive chemotaxis toward leukotriene B 4 (LTB 4 ) as determined by a microfluidic μC 3 assay. (B) Percentage of migrated neutrophils toward LTB 4 . (C) Representative micrographs showing directional (straight channels) vs. non-directional migration (cells were lost within the maze). Data obtained from 12-week-old mice, n = 5/group.
*
➢ How do QACs disinfectants affect the systemic immune dysregulation in lupus ? ❑ Research Question ➢ Delineate the effects of ambient exposure to QACs disinfectants on the progression of lupus-like disease in the MRL/lpr mouse model. ❑ Research objectives ❑ Background ✓ Hypersensitivity reactions and immune dysregulation have been reported with the use of quaternary ammonium compound disinfectants (QACs).
40
50
15
*
**
40
30
10 %Of CD3 + 20
10 %Of CD3 + 20 30
10
5
Decreased Infiltration of Neutrophils “Into the Spleen”
0
0
0
Annexin V + PI + T
Annexin V + PI - T
EM cells
CM cells
T EM cells
Early/Late apoptotic
(A)
(D)
(E)
(F)
*
*
150
10 12 14 16 18
100
30
(B)
(C)
80
***
100
80
10 % Of CD3 + 20
500
*
60
0 2 4 6 8
400
40
60
50
20
300
IL6(pg/ml)
40
IL6:18srRNA
0
0
0
20 TNF (pg/ml)
200
Annexin V + PI + DN T cells
Annexin V + PI - DN T cells
Splenic expression
Serum Level
100
Figure 8. Increased apoptosis of T cells with ambient exposure to QACs. (A) Percentage of early apoptotic TCM cells gated as Annexin V+ PI− on CD3+CD62L+CD44+ cells. (B) Percentage of late apoptotic or necrotic TEM cells gated as Annexin V+ PI+ on CD3+CD62L-CD44+ cells. (C) Ratio of early apoptotic (Annexin V+ PI−) to late apoptotic or necrotic (Annexin V+ PI+) TEM 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 1 st experimental setting.
0 Mean Intensity
0
Ly6G
Figure.1 Lupus is multi-system autoimmune disorder with very diverse clinical manifestations that are strongly influenced by environmental factors.
(D)
(E)
Figure 5. Decreased infiltration of neutrophils into the spleen with ambient exposure to QACs.(A) Representative micrographs of Immunohistochemistry (IHC)-stained splenic sections showing Ly6G+ neutrophils.(B) Quantification of the micrographs shown as the mean intensity of PE fluorescence.(C) Serum level of TNF α . (D)Transcript level of CXCR4 in Bone marrow-enriched neutrophils. (E) Ratio of the transcript levels of CXCR4 and CXCR2 .
**
*
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Neutrophilic expression
10
❑ Study design
CONCLUSION & FUTURE DIRECTIONS
Δ CXCR4/ Δ CXCR2
5
Figure 2. Experimental design showing two experimental settings. (A) We compared the F2 generation of QAC-free MRL/lpr mice (were bred and maintained in the IDU facility with no use of QACs) with age-matched QAC- exposed mice. These QAC- exposed mice were bred in a facility with minimal QAC use (Phase IV) then were transferred to the QAC-use facility (ILSB) at weaning. (B)We compared the F4 generation of QAC-free MRL/lpr siblings where some were transferred to the QAC-use facility (ILSB) at weaning that constituted the QAC-exposed group.
(A)
Figure 9. Ambient exposure to QACs disinfectant could alter immune cell phenotypes, functions, and their fate. Ambient exposure of lupus-prone mice to QACs disinfectants reduced lupus- associated splenomegaly. QACs exposures likely mediated neutrophil toning of T cell responses. As evident by hindering neutrophil migration and upregulating their PD- L1 signaling which in turn toned T cell activation and induced their apoptosis . ❑ Future directions ➢ How do QACs affect immunity under steady-state conditions?
0
Neutrophilic expression CXCR4:18srRNA
Upregulated PD-1/PD-L1 Expression
(A)
(B)
500
****
400
300
200
(B)
100
0 Mean Intensity
(C)
PD-1
800
***
600
400
200
0 Mean Intensity
ACKNOWLEDGMENTS
PD-L1
❑ Statistical Analysis Student’s t test was employed for the comparison between groups. All data are represented as mean ± SEM. ≠P < 0.10, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 . All analyses were performed with Prism GraphPad.
Figure 6. Upregulated PD-1/PD-L1 expression with Ambient Exposure to QACs. (A) Representative micrographs of IHC-stained splenic sections showing the co-localization of PD-1 (green) with PD-L1 (blue). Pictures were captured with a Zeiss LSM880 confocal microscope. (B – C) Quantification of the mean intensity of PD-1 (B) and PD-L1 (C) fluorescence in IHC-stained splenic sections.
113 2 0 2 1 R e s e a r c h R e c o g n i t i o n D a y
Made with FlippingBook flipbook maker