Immune cell expression patterns of CD39/CD73 ectonucleotidases in rodent models of cardiac arrest and resuscitation

Abstract

Background: Cardiac arrest (CA) is a significant public health concern. There is the high imminent mortality and survival in those who are resuscitated is substantively compromised by the post-CA syndrome (PCAS), characterized by multiorgan ischemia-reperfusion injury (IRI). The inflammatory response in PCAS is complex and involves various immune cell types, including lymphocytes and myeloid cells that have been shown to exacerbate organ IRI, such as myocardial infarction. Purinergic signaling, as regulated by CD39 and CD73, has emerged as centrally important in the context of organ-specific IRI. Hence, comprehensive understanding of such purinergic responses may be likewise imperative for improving outcomes in PCAS.

Methods: We have investigated alterations of immune cell populations after CA by utilizing rodent models of PCAS. Blood and spleen were collected after CA and resuscitation and underwent flow cytometry analysis to evaluate shifts in CD3⁺CD4⁺ helper T cells, CD3⁺CD8a⁺ cytotoxic T cells, and CD4/CD8a ratios. We then examined the expression of CD39 and CD73 across diverse cell types, including myeloid cells, T lymphocytes, and B lymphocytes.

Results: In both rat and mouse models, there were significant increases in the frequency of CD3⁺CD4⁺ T lymphocytes in PCAS (rat, P < 0.01; mouse, P < 0.001), with consequently elevated CD4/CD8a ratios in whole blood (both, P < 0.001). Moreover, CD39 and CD73 expression on blood leukocytes were markedly increased (rat, P < 0.05; mouse, P < 0.01 at 24h). Further analysis in the experimental mouse model revealed that CD11b⁺ myeloid cells, with significant increase in their population (P < 0.01), had high level of CD39 (88.80 ± 2.05 %) and increased expression of CD73 (P < 0.05). CD19⁺ B lymphocytes showed slight increases of CD39 (P < 0.05 at 2h) and CD73 (P < 0.05 at 2h), while, CD3⁺ T lymphocytes had decreased levels of them. These findings suggested a distinct patterns of expression of CD39 and CD73 in these specific immune cell populations after CA.

Conclusions: These data have provided comprehensive insights into the immune response after CA, highlighting high-level expressions of CD39 and CD73 in myeloid cells.

Keywords: B cells; T cells; cardiopulmonary resuscitation; heart arrest; ischemia; monocytes; reperfusion injury; rodent.

Figure 1

Experimental protocol on animal surgery. (A) Animal protocol of rat CA. Rats were subjected to 12 minutes of asphyxia-induced CA and resuscitation. Following observation under mechanical ventilation for 120 minutes after initiating CPR, whole blood was collected. (B) Animal protocol of mouse CA. Mice were subjected to 8 minutes of KCl-induced CA and resuscitation. At 2 hours or 24 hours after initiating CPR, whole blood and the spleen were collected.

Figure 2

CD3, CD4, and CD8a expression in whole blood of rat CA model. (A) Representative images of dot plots through gating process. Lymphocytes were gated. (B) Representative dot plot images of CD3, CD4, and CD8a positive leukocytes. (C) CD3⁺CD4⁺ ratio in lymphocytes population. (D) CD3⁺CD8a⁺ ratio in lymphocytes population. (E) CD4/CD8a ratio. Data were expressed as means ± SEM (n = 9 rats/group). Two groups were compared by Wilcoxon matched-pairs signed rank test (** P < 0.01).

Figure 3

CD3, CD4, and CD8a expression in whole blood of mouse CA model. (A) Representative images of dot plots through gating process. Lymphocytes were gated. (B) Representative dot plot images of CD3, CD4, and CD8a positive leukocytes. (C) CD3⁺CD4⁺ ratio in lymphocytes population. (D) CD3⁺CD8a⁺ ratio in lymphocytes population. (E) CD4/CD8a ratio. Data were expressed as means ± SEM (n = 8 mice/group). Two groups were compared by Mann-Whitney test (** P < 0.01, *** P < 0.001).

Figure 4

CD3, CD4, and CD8a expression in the spleen of mouse CA model. (A) Representative images of dot plots through gating process. Lymphocytes were gated. (B) Representative dot plot images of CD3, CD4, and CD8a positive leukocytes. (C) CD3⁺CD4⁺ ratio in lymphocytes population. (D) CD3⁺CD8a⁺ ratio in lymphocytes population. (E) CD4/CD8a ratio. Data were expressed as means ± SEM (n = 8 mice/group). Two groups were compared by Mann-Whitney test (* P < 0.05).

Figure 5

CD39 and CD73 expression in whole blood of rat CA model. (A) Representative images of dot plots through gating process. Leukocytes were gated. (B) Representative overlaid histograms of the CD39 fluorescence intensity. (C) CD39 median fluorescence intensity. (D) Representative overlaid histograms of CD73 fluorescence intensity. (E) CD73 median fluorescence intensity. Data were expressed as means ± SEM (n = 5 rats/group). Two groups were compared by Wilcoxon matched-pairs signed rank test (* P < 0.05).

Figure 6

CD39 and CD73 expression in CD45⁺ leukocytes from whole blood and the spleen of mouse CA model. (A) Representative images of dot plots through gating process. CD45⁺DAPI^- leukocytes were gated. (B) Representative dot plot images of CD39, CD73, isotype controls, and CD45. (C) CD45⁺CD39⁺ ratio and (D) CD45⁺CD73⁺ ratio in CD45⁺ leukocytes in whole blood. (E) CD45⁺CD39⁺ ratio and (F) CD45⁺CD73⁺ ratio in CD45⁺ leukocytes in the spleen. Data were expressed as means ± SEM (n = 3-6 mice/group). Between naïve and 2 hours or 24 hours after CA groups, two groups were compared by Dunn’s multiple comparisons test (* P < 0.05, ** P < 0.01).

Figure 7

CD39 and CD73 expression in CD11b⁺ myeloid cells from whole blood and the spleen of mouse CA model. (A) Representative images of dot plots through gating process. CD45⁺DAPI^- leukocytes were gated. (B) Representative histogram images of CD11b in CD45⁺ leukocytes. CD11b⁺ myeloid cells were gated. CD11b⁺ ratio in CD45⁺ leukocytes. (C) Representative dot plot images of CD39, CD73, isotype controls, and CD11b. (D) CD11b⁺CD39⁺ ratio and (E) CD11b⁺CD73⁺ ratio in CD11b⁺ myeloid cells in whole blood. (F) CD11b⁺CD39⁺ ratio and (G) CD11b⁺CD73⁺ ratio in CD11b⁺ myeloid cells in the spleen. Data were expressed as means ± SEM (n = 3-6 mice/group). Between naïve and 2 hours or 24 hours after CA groups, two groups were compared by Dunn’s multiple comparisons test (* P < 0.05, ** P < 0.01).

Figure 8

CD39 and CD73 expression in CD3⁺ T lymphocytes from whole blood and the spleen of mouse CA model. (A) Representative images of dot plots through gating process. DAPI^- leukocytes were gated. (B) Representative dot plot images of CD19 and CD3 in leukocytes. CD3⁺ T lymphocytes were gated. CD3⁺ ratio in leukocytes. (C) Representative dot plot images of CD39, CD73, isotype controls, and CD3. (D) CD3⁺CD39⁺ ratio and (E) CD3⁺CD73⁺ ratio in CD3⁺ T lymphocytes in whole blood. (F) CD3⁺CD39⁺ ratio and (G) CD3⁺CD73⁺ ratio in CD3⁺ T lymphocytes in the spleen. Data were expressed as means ± SEM (n = 3-6 mice/group). Between naïve and 2 hours or 24 hours after CA groups, two groups were compared by Dunn’s multiple comparisons test (** P < 0.01).

Figure 9

CD39 and CD73 expression in CD19⁺ B lymphocytes from whole blood and the spleen of mouse CA model. (A) Representative images of dot plots through gating process. DAPI^- leukocytes were gated. (B) Representative dot plot images of CD19 and CD3 in leukocytes. CD19⁺ B lymphocytes were gated. CD19⁺ ratio in leukocytes. (C) Representative dot plot images of CD39, CD73, isotype controls, and CD19. (D) CD19⁺CD39⁺ ratio and (E) CD19⁺CD73⁺ ratio in CD19⁺ B lymphocytes in whole blood. (F) CD19⁺CD39⁺ ratio and (G) CD19⁺CD73⁺ ratio in CD19⁺ B lymphocytes in the spleen. Data were expressed as means ± SEM (n = 3-6 mice/group). Between naïve and 2 hours or 24 hours after CA groups, two groups were compared by Dunn’s multiple comparisons test (* P < 0.05, ** P < 0.01).