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Differential lipid signaling from CD4+ and CD8+ T cells contributes to type 1 diabetes development


. 2024 Sep 18:15:1444639.


doi: 10.3389/fimmu.2024.1444639.


eCollection 2024.

Affiliations

Item in Clipboard

Tayleur D White et al.


Front Immunol.


.

Abstract


Introduction:

We reported that Ca2+-independent phospholipase A2β (iPLA2β)-derived lipids (iDLs) contribute to type 1 diabetes (T1D) onset. As CD4+ and CD8+ T cells are critical in promoting β-cell death, we tested the hypothesis that iDL signaling from these cells participates in T1D development.


Methods:

CD4+ and CD8+ T cells from wild-type non-obese diabetic (NOD) and NOD.iPLA2β+/- (NOD.HET) mice were administered in different combinations to immunodeficient NOD.scid.


Results:

In mice receiving only NOD T cells, T1D onset was rapid (5 weeks), incidence 100% by 20 weeks, and islets absent. In contrast, onset was delayed 1 week and incidence reduced 40%-50% in mice receiving combinations that included NOD.HET T cells. Consistently, islets from these non-diabetic mice were devoid of infiltrate and contained insulin-positive β-cells. Reduced iPLA2β led to decreased production of proinflammatory lipids from CD4+ T cells including prostaglandins and dihydroxyeicosatrienoic acids (DHETs), products of soluble epoxide hydrolase (sEH), and inhibition of their signaling decreased (by 82%) IFNγ+CD4+ cells abundance. However, only DHETs production was reduced from CD8+ T cells and was accompanied by decreases in sEH and granzyme B.


Discussion:

These findings suggest that differential select iDL signaling in CD4+ and CD8+ T cells contributes to T1D development, and that therapeutics targeting such signaling might be considered to counter T1D.


Keywords:

T-lymphocytes; adoptive transfer; flow cytometry; islet microscopy; lipid signaling; lipidomics; type 1 diabetes.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures


Figure 1



Figure 1

Splenocytes adoptive transfer. (A) Comparison of iPla2β mRNA in NOD males and females. Splenocytes were prepared from 8- to 10-week-old wild-type NOD mice and processed for qPCR analysis of iPla2β mRNA. Fold-change relative to male NOD are presented as mean ± SEM (n = 3 in each group). (B) Genotyping. DNA was generated from tail clips and progeny were genotyped by PCR analyses. Reactions were performed in the presence of primers for the NOD sequence or for the disrupted sequence (NOD.iPLA2β+/-) for each mouse. Representative expected bands for the wild-type NOD (1400 bp), NOD.iPLA2
β+/- (NOD.HET, 1400 and 400 bp), and NOD.iPLA2
β-/- (NOD.KO, 400 bp) (n = 2 in each group). (C)
iPla2β mRNA in the NOD models. Splenocytes were prepared from 8- to 10-week-old female mice and processed for qPCR analyses. Fold-changes relative to NOD are presented as mean ± SEM (NOD, n = 6, NOD.HET, n = 4; NOD.KO, n = 5). (D) Experimental design. (E) Diabetes incidence. NOD.scid mice were administered splenocytes (i.p., 2.5 × 106 cells/mouse in 70 μL of PBS) prepared from NOD, NOD.HET, and NOD.KO. Blood glucose was monitored weekly and T1D onset was recorded upon two consecutive readings >275 mg/dl. (p-values, NOD vs. NOD.HET and NOD vs. NOD.KO are indicated.) (F–H) Intraperitoneal glucose tolerance testing (IPGTT). Mice were fasted overnight before obtaining fasting blood glucose levels. The mice were then administered glucose 2 g/kg body weight and blood glucose in a 2 µL aliquot of tail vein blood was measured over a 2 h-period. (I) Area under the curve. Data obtained through the IPGTT were used to calculate the area under the curve, as an index of glucose tolerance.


Figure 2



Figure 2

Islet infiltration and insulin staining. NOD mice were treated as described in
Figure 1
. (A, C, D) Hematoxylin/eosin (H/E) staining. Paraffin sections (6 μm) of pancreas were prepared (at onset of diabetes or at 30 weeks of age) from the NOD, NOD.HET, and NOD.KO groups and stained with H/E. Representative images from three mice in each group are presented. H/E-stained pancreas magnification is 4× and individual islet magnification is 40×. Scale bar of pancreas is 20 μm and islets scale bar is 50 μm. (B, F, G) Insulin staining. Paraffin sections (6 μm) were stained for insulin (green) and nucleus (DAPI, blue). Representative images of islets from three mice in each group are presented. Insulin-stained pancreas magnification is 4× and individual islet magnification is 40×. Scale bar for insulin-stained pancreas is 1 mm and individual islets 50 μm. (E) Insulitis. Percent infiltration for each islet was calculated as the value of noninfiltrated area subtracted from total islet area [% infiltrate = 100 × (total area–non-infiltrated area)/total area] using ImageJ software. Data are mean ± SEM of percent of islet infiltrated. NOD, NOD.HET, and NOD.KO groups were analyzed (n = 3 animals from each group; islets in each NOD = 0, NOD.HET = 4, 8, 10; and NOD.KO = 10, 2, 10. (H) β-cell area. Dividing the insulin-stained area, representing β cells, by total pancreas area: % β cells = 100 × [β-cell area sum per pancreas/pancreas area total]. Data are presented as mean ± SEM (NOD (n = 5), NOD.HET (n = 6), and NOD.KO (n = 6); islets in each NOD = 0 in each; NOD.HET = 1, 1, 2, 16, 37, and 2; and NOD.KO = 13, 20, 7, 25, 2 and 0. [(E, H) NID = no islets detected].


Figure 3



Figure 3

Splenocyte Immune Cell Composition. Age-matched NOD, NOD.HET, and NOD.KO female spleen were processed for flow cytometry to analyze the splenic immune cell composition. Analyses were done at 4, 8, and 14 weeks. (A) CD4+ T cells relative to CD45+. (C) Naïve CD4+ T cells relative to total CD4+ population. (E) Activated CD4+ T cells relative to total CD4+ population. (B) CD8+ T cells relative to CD45+. (D) Naïve CD8+ T cells relative to total CD8+ population. (F) Activated CD8+ T cells relative to total CD8+ population. Surface markers utilized included CD45, CD4, CD8, CD62L, and CD44. NOD: 4 weeks (n = 4), 8 weeks (n = 7), and 14 weeks (n = 11). NOD.HET: 4 weeks (n = 8), 8 weeks (n = 8), and 14 weeks (n = 14). NOD.KO: 4 weeks (n = 3), 8 weeks (n = 8), and 14 weeks (n = 8).


Figure 4



Figure 4

T-cell adoptive transfer. (A) Verification of T-cell purity. Splenic T cells were prepared using StemCell positive and negative selection columns and purity assessed by flow analyses using markers for CD4+ and CD8+ cells (
Supplementary Figure S1
). CD4+ cells and CD8+ T cells are represented in quadrants 1 and 3, respectively. (B)
iPla2
β mRNA. Purified T cells 10- to 12-week-old from male and female NOD were processed for qPCR analyses. Fold-change relative to NOD are presented as mean ± SEM (p-value of male vs. female indicated, n = 3 in each group). (C) Splenic T-cell–iPla2
β mRNA. Purified T cells were processed for qPCR analyses. Fold-changes relative to NOD are presented as mean ± SEM (p-values of NOD vs. NOD.HET indicated, n = 3 in each group). (D) Experimental design. (E) Diabetes incidence. NOD.scid (5-week-old) were administered (i.p.) CD4+/CD8+ T cells in a 3:1 ratio (7.5 × 106 CD4+: 2.5 × 106 CD8+) and blood glucose was monitored weekly and T1D onset recorded, as described in
Figure 1
. (p-values of NOD CD4 + HET CD8 or HET CD4 + NOD CD8 vs. NOD CD4 + CD8 are indicated.) (F) Area under the curve. Data obtained through the IPGTT were used to calculate the area under the curve, as an index of glucose tolerance.


Figure 5



Figure 5

Islet infiltration and insulin staining. Mice were treated as described in
Figure 3
. (A, C, D) Hematoxylin/eosin (H/E) staining. Paraffin sections (6 μm) of pancreas were prepared (at T1D onset or at 30 weeks of age) from NOD.scid recipients of NOD CD4+ T cells + NOD CD8+, NOD CD4+ + NOD.HET CD8+ or NOD.HET CD4+ + NOD CD8+ T cells and stained with H/E. Representative images from three mice in each group are presented. H/E-stained pancreas magnification is 4× and individual islet magnification is 40×. Scale bar of H/E-stained pancreas is 1 mm and individual islets are 50 μm. (B, F, G) Insulin staining. Paraffin sections (6 μm) of pancreas were stained for insulin (green) and nucleus (DAPI, blue). Representative images of islets from three mice in each group are presented. Insulin-stained pancreas magnification is 4× and individual islet magnification is 40×. Scale bar of insulin-stained pancreas is 500 μm and individual islets are 50 μm. (E) Insulitis. Percent infiltration for each islet was calculated as the value of non-infiltrated area subtracted from total islet area [% infiltrate = 100 × (total area − non-infiltrated area)/total area] using ImageJ software. Data are presented as mean ± SEM (three animals in each group; islets in each NOD CD4+
+ NOD CD8+ = 0 in each; NOD.HET CD4+
+ NOD CD8+ = 10, 1, and 7 (n = 18); and NOD CD4+
+ NOD.HET CD8+ = 8, 2, and 10 (n = 20). (H) β-cell area. Determined by dividing the insulin-stained area, representing β cells, by total pancreas area: % β cells = 100 × (β-cell area sum per pancreas/pancreas area total). Data are presented as mean ± SEM (n = 3 animals in each group; islets in each NOD CD4+
+ NOD CD8+= 0 in each; NOD.HET CD4+
+ NOD CD8+ = 10, 1, and 7, and NOD CD4+
+ NOD.HET CD8+ = 8, 2, and 10. (E and H, NID = no islets detected). (I) Circulating Insulin levels. Plasma was collected from diabetic (D)
NOD CD4+ + NOD CD8+ (n = 9), NOD CD4+ + NOD.HET CD8+ (n = 3), NOD.HET CD4+ + NOD CD8+ (n = 4) and non-diabetic (ND) NOD CD4+ + NOD.HET CD8+ (n = 3), NOD.HET CD4+ + NOD CD8+ (n = 4) mice and circulating insulin levels were determined by ELISA. (a
NOD CD4+ + NOD CD8+ D vs. NOD.HET CD4+
+ NOD CD8+ ND, p < 0.005; b
NOD CD4+ + NOD.HET CD8+ D vs. NOD.HET CD4+ + NOD CD8+ ND, p < 0.01; c
NOD CD4+ + NOD.HET CD8+ D vs. NOD CD4+ + NOD.HET CD8+ ND, p < 0.005; d
NOD CD4+ + NOD.HET CD8+ ND vs. NOD.HET CD4+ + NOD CD8+ D, p < 0.005; eNOD.HET CD4+ + NOD CD8+ D vs. NOD.HET CD4+ + NOD CD8+ ND, p < 0.005; and f
NOD CD4+ + NOD CD8+ D vs. NOD CD4+ + NOD.HET CD8+ ND, p < 0.005).


Figure 6



Figure 6

Ex vivo analyses of CD4+ T cells. (A) Lipidomics. CD4+ T cells were purified from 14-week-old NOD and NOD.HET and cultured for 48 h under basal conditions. The media was then collected for lipidomics analyses. (p-values, NOD vs. NOD.HET indicated.) (B, C) IFNγ production by CD4+ T cells. CD4+ T cells from NOD were differentiated to Th1 cells and cultured for 24 h. They were then pretreated with DMSO alone (Con), grapiprant (G, 1 μM), or TPPU (T, 10 μM) for 2 h prior to stimulation with ionomycin (670 μM) + PMA (40.5 μM) for an additional 24 h. The cells were then subjected to flow analyses to quantitate IFNγ+CD4+ T cells. The CD4+ T cells from NOD.HET were similarly differentiated and subsequently stimulated in the absence or presence of PGE2 (P, 1 μM). (B) Flow analyses demonstrating gating on IFNγ+CD4+ T cells under each condition. (C) Fold-changes in IFNγ+CD4+ T cells relative to DMSO are presented as mean ± SEM (NOD, n = 12; T, n = 12; NOD + T, n = 12; G (n = 6); NOD + G, n = 6; NOD.HET, n = 4; NOD.HET + P, n = 4; and NOD.HET + P + stimulation, n = 3. (p-values between groups indicated).


Figure 7



Figure 7

Ex vivo analyses of CD8+ T cells. (A) Lipidomics. CD8+ T cells were purified from 14-week-old NOD and NOD.HET and cultured for 24 h in the presence of hIL-2 (30 U/mL) and IL-7 (0.50 ng/mL). The cells were then stimulated using ebioscience stimulation cocktail containing ionomycin and PMA for 4 h. The cells were then collected for flow and media for lipidomics analyses. (p-value, NOD vs. NOD.HET indicated.) (B) IFNγ+CD8+ analyses. CD8+ T cells were treated with DMSO alone, grapiprant (G, 1 μM), or TPPU (T, 10 μM) in combination with ionomycin (670 μM) + PMA (40.5 μM) for 4 h. NOD.HET were similarly stimulated in the absence or presence of PGE2 (P, 1 μM). Fold changes in IFNγ+CD8+ T cells relative to DMSO are presented as mean ± SEM. (NOD, n = 6; T, n = 6; NOD + T, n = 6; G (n = 6); NOD + G, n = 6; NOD.HET, n = 7; NOD.HET + P, n = 7; and NOD.HET + P + stimulation, n = 7). (C–E) CD8+ T-cell mRNA analyses. Purified CD8+ T cells were processed for qPCR analyses of perforin, sEh, and granzyme B mRNA. Fold changes relative to NOD are presented as mean ± SEM (NOD, n = 3–7; NOD.HET, n = 3–6). (p-values, NOD vs. NOD.HET indicated).


Figure 8



Figure 8

Proposed model of CD4+ and CD8 + T-cell–iPLA2β in T1D development. We suggest that iPLA2β in T cells promotes the production of inflammatory lipids (i.e., prostaglandins, DHETs, and leukotrienes), secretion of proinflammatory cytokines (i.e., IFNγ and TNFα), and granzyme B expression to promote T1D development. However, these outcomes are mitigated with reduced iPLA2β resulting in lowering T1D onset.

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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by funding from R01 DK110292, R21 AI169214-01, JDRF 2-SRA-2022-1210-S-B, JDRF RFA 1-INO-2023-1344-A-N, UAB Department of CDIB, UAB Comprehensive Diabetes Center, and UAB-DRC to SR; R01DK069455 Diversity Research Supplement, UAB Diversity Research Supplement and T32 GM008111 to TDW; partial support was provided by NIH/NIEHS (RIVER Award) R35 ES030443-01 and NIH/NIEHS (Superfund Award) P42 ES004699 to BH; and The Veteran’s Administration (VA Merit Reviews, BX001792 and BX006063) (CEC), Research Career Scientist Award (IK6BX004603), the National Institutes of Health by way of R01s AI139072, DK126444, and GM137578 to CC.


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