Methamphetamine administration targets multiple immune subsets and induces phenotypic alterations suggestive of immunosuppression

Abstract

Methamphetamine (Meth) is a widely abused stimulant and its users are at increased risk for multiple infectious diseases. To determine the impact of meth on the immune system, we utilized a murine model that simulates the process of meth consumption in a typical addict. Our phenotypic analysis of leukocytes from this dose escalation model revealed that meth affected key immune subsets. Meth administration led to a decrease in abundance of natural killer (NK) cells and the remaining NK cells possessed a phenotype suggesting reduced responsiveness. Dendritic cells (DCs) and Gr-1(high) monocytes/macrophages were also decreased in abundance while Gr-1(low) monocytes/macrophages appear to show signs of perturbation. CD4 and CD8 T cell subsets were affected by methamphetamine, both showing a reduction in antigen-experienced subsets. CD4 T cells also exhibited signs of activation, with increased expression of CD150 on CD226-expressing cells and an expansion of KLRG1(+), FoxP3(-) cells. These results exhibit that meth has the ability to disrupt immune homeostasis and impact key subsets of leukocytes which may leave users more vulnerable to pathogens.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Gating strategy for defining major subsets of leukocytes among splenocytes. A.

Dead cells were removed from the analysis using Live/Dead® fixable dead cell stain. B. Doublets were removed from living cells (Live/Dead−) using FSC-A and FSC-H. C. Gr-1bright splenocytes were gated and defined as neutrophils. D. NKp46+ events were gated from Gr-1low/− splenocytes and defined as natural killer (NK) cells. E. NKp46−, Gr-1low/− splenocytes were divided into MHC II+ and MHC II− populations. F. MHC II+ events were split by B220 and CD11c expression. MHC II+, B220+, CD11c− events were defined as B cells. MHC II+ B220−, CD11c+ events were defined as dendritic cells (DCs). G. We split MHC II− events by CD3 and CD11b expression. CD11b-, CD3+, MHC II- events were labeled as T cells. H. CD11b+, CD3−, MHC II- events were split according to SSC profile. Events with higher SSC values (suggesting greater internal granularity) were labeled as eosinophils. I. Events with lower SSC values were divided by Gr-1 expression, giving two populations: Gr-1(Ly-6C)high monocytes/macrophages and Gr-1(Ly-6C)low monocytes/macrophages (mono/MΦ).

Figure 2. Meth causes a reduction of splenic leukocyte subsets.

C57BL/6 mice were treated with vehicle (control) or ramped dosage of methamphetamine (meth – see methods) for 14 days. At day 14, mice were sacrificed and splenic leukocyte subsets were analyzed by flow cytometry. A. Meth treatment did not alter total splenocyte number. B. Meth causes a significant reduction in total natural killer (NK) cells (NKp46+, CD3−, B220−, Gr-1−, MHC II−). C. Gr-1high monocytes (CD11b+, Gr-1high, MHC II−, CD3−, B220−) were reduced after meth treatment. D. Meth treatment reduces dendritic cells (DCs) defined as CD11c+, MHC II+, B220−, Gr-1− events. Dead cells and doublets were removed prior to analysis. **P<0.01 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 3. Meth perturbs Gr-1low monocytes/macrophages.

Gr-1low monocytes/macrophages were analyzed for signs of activation after meth treatment using flow cytometry. A. Representative histogram showing CD80 (B7-1) expression by Gr-1ow monocytes/macrophages. Red represents a meth-treated animal, black represents a vehicle-treated animal, and grey is the isotype control. B. Meth causes increased expression of CD80 by Gr-1low, quantified by mean fluorescence intensity (MFI). C. Representative histogram showing CD11b (Mac-1) expression by Gr-1low monocytes/macrophages with colors as in A above. D. Meth causes decreased expression of CD11b by Gr-1low monocytes, quantified by MFI. Dead cells and doublets were removed prior to analysis. **p<0.01, ***p<0.001 calculated by Mann-Whitney U Test. Error bars represent SEM. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 4. Meth alters NK cell subsets.

We labeled splenic NK cells (NK1.1+, CD4−, CD8−) with antibodies against killer cell lectin-like receptor G1 (KLRG1) and CD27 to determine if meth treatment alters functionally distinct NK cell subsets. A. Representative gating showing KLRG1 and CD27 on gated NK cells. B and C. Meth treatment results in a decreased proportion of KLRG1−, CD27+ NK cells with an increase in proportion of KLRG1+, CD27− NKs. D and E. Although proportionally higher, KLRG1+, CD27− NK cells are not increased in absolute number. However, KLRG1−, CD27+ NK cells were significantly reduced in number. Dead cells and doublets were removed prior to analysis. ***p<0.001 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 5. Meth reduces proportions of CD62Llow, CD44high splenic CD4 T cells and these cells exhibit lower CD27 expression.

Splenic CD4 T cells were examined after meth treatment to determine if meth alters surface phenotypes suggesting activation/antigen experience and effector status. A. Representative gating showing CD62L and CD44 on CD4 T cells. B. Meth causes a reduction in proportion of CD44high, CD62Llow CD4 T cells. C. CD27 expression by CD44high, CD62Llow CD4 T cells. D. CD44high, CD62Llow CD4 T cells from meth treated animals exhibit a lower proportion of cells expressing CD27 at a high level. **p<0.01, ***p<0.001 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 6. Meth causes reduced proportions of alternatively spliced CD4 T cells among the splenic CD62Lhigh compartment.

Meth and control mice were analyzed for surface expression of CD45RB and CD44 on the CD62Lhigh subset. CD45RB expression is downregulated upon antigen experience and can be used to gauge activation levels of CD4 T cells. A. CD45RB and CD44 on CD62Lhigh splenic CD4 T cells. B–D. Meth-treated animals had a higher proportion of CD45RBhigh, CD44low CD4 T cells compared to controls. Meth-treated animals also exhibited lower proportions of CD45RBlow, CD44low and CD45RBlow, CD44highsubsets compared to controls. ***p<0.001 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 7. Meth treatment causes a reduction in proportion of CD62Llow, CD44high CD4 T cells in the mesenteric lymph node (MLN).

MLN CD4 T cells were labeled with CD62L and CD44 to determine proportions of antigen-experienced cells. A. CD62L and CD44 on MLN CD4 T cells from meth and vehicle treated animals. B. Meth causes a reduction in proportion of CD44high, CD62Llow CD4 T cells in the MLN. **p<0.01 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 8. Increased expression of CD150 on CD226+ CD4 T cells after meth treatment.

We examined the expression of the costimulatory markers CD150 and CD226 by splenic CD4 T cells after meth treatment. A. Representative gating and histogram showing selection of CD226+ CD4 T cells and increased expression of CD150 by meth treated animals compared to control. Red represents a meth-treated animal, black is vehicle-treated animal, and grey is the isotype control. B. CD226+ CD4 T cells from meth treated animals express higher levels of CD150 than those from control animals. Protein expression was calculated using mean fluorescence intensity (MFI). ***p<0.001 calculated by Mann-Whitney U Test. Error bars represent SEM. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 9. Meth promotes the expansion of splenic KLRG1+, FoxP3− CD4 T cells.

Meth-treated animals possessed an expansion of KLRG1+ CD4 T cells within the spleen. These cells were CD44high and negative/low for FoxP3, CD25, CD62L, NKG2D, and CD45RB. A. Representative gating showing significant expansion of splenic KLRG1+, FoxP3− CD4 T cells among the CD44high, CD45RBlow compartment with relatively unchanged proportions of FoxP3-expressing subsets after meth treatment. B and C. Proportion and absolute number of KLRG1+, FoxP3− CD4 cells are increased with meth. ***p<0.001 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 10. Meth-treated animals exhibit a greater proportion of naïve phenotype CD8 T cells and reduced proportions of antigen-experienced subsets within the spleen.

Splenic CD8 T cells were labeled with CD62L and CD44 and classified into 4 groups according the expression of these two proteins: CD62Lhigh, CD44low (Naïve CD8 T cell, TN), CD62Lhigh, CD44high, (central memory CD8 T cell, TCM), CD62Llow, CD44high (effector memory CD8 T cells, TEM), and CD62Llow, CD44low (acute effector CD8 T cell, TAE). A. Gating showing CD44 and CD62L expression by splenic CD8 T cells. B–E. After meth treatment, the TN compartment is increased in proportion, while the TAE and TEM showed a decrease in proportion and TCM appeared unchanged. *p<0.05, **p<0.01 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 11. CD8 T cells from the mesenteric lymph node (MLN) also show reductions in proportion of antigen-experienced subsets with an increase in the naïve compartment.

Mesenteric lymph node CD8 T cells were labeled with CD62L and CD44 and classified as in Fig. 10. A. Gating showing CD44 and CD62L expression by MLN CD8 T cells. B–E. After meth treatment, the TN compartment is increased in proportion, while the TAE, TEM, and TCM compartments all revealed a decrease in proportion. **p<0.01, ***p<0.001 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 12. Meth causes a reduced proportion and number of KLGR1+ CD8 T cells in the MLN.

CD8 T cells were labeled with KLRG1 to investigate whether meth impacts subsets of antigen-experienced CD8 T cells. A. The proportion and number of CD8 T cells expressing KLRG1 in the MLN is reduced after meth treatment. *p<0.01, ***p<0.001 calculated by Mann-Whitney U Test. Bars represent mean. Data are from 2 experiments of 5 animals per treatment group per experiment.

Figure 13. Methamphetamine impacts the innate and adaptive arms of immunity.

Summary of the effects of methamphetamine on immune cell subsets based upon the observations in this report.