Microenvironmental regulation of T-cells in pulmonary hypertension

Abstract

Introduction: In pulmonary hypertension (PH), pulmonary arterial remodeling is often accompanied by perivascular inflammation. The inflammation is characterized by the accumulation of activated macrophages and lymphocytes within the adventitial stroma, which is comprised primarily of fibroblasts. The well-known ability of fibroblasts to secrete interleukins and chemokines has previously been implicated as contributing to this tissue-specific inflammation in PH vessels. We were interested if pulmonary fibroblasts from PH arteries contribute to microenvironmental changes that could activate and polarize T-cells in PH.

Methods: We used single-cell RNA sequencing of intact bovine distal pulmonary arteries (dPAs) from PH and control animals and flow cytometry, mRNA expression analysis, and respirometry analysis of blood-derived bovine/human T-cells exposed to conditioned media obtained from pulmonary fibroblasts of PH/control animals and IPAH/control patients (CM-(h)PH Fibs vs CM-(h)CO Fibs).

Results: Single-cell RNA sequencing of intact bovine dPAs from PH and control animals revealed a pro-inflammatory phenotype of CD4+ T-cells and simultaneous absence of regulatory T-cells (FoxP3+ Tregs). By exposing T-cells to CM-(h)PH Fibs we stimulated their proinflammatory differentiation documented by increased IFNγ and decreased IL4, IL10, and TGFβ mRNA and protein expression. Interestingly, we demonstrated a reduction in the number of suppressive T-cell subsets, i.e., human/bovine Tregs and bovine γδ T-cells treated with CM-(h)PH-Fibs. We also noted inhibition of anti-inflammatory cytokine expression (IL10, TGFβ, IL4). Pro-inflammatory polarization of bovine T-cells exposed to CM-PH Fibs correlated with metabolic shift to glycolysis and lactate production with increased prooxidant intracellular status as well as increased proliferation of T-cells. To determine whether metabolic reprogramming of PH-Fibs was directly contributing to the effects of PH-Fibs conditioned media on T-cell polarization, we treated PH-Fibs with the HDAC inhibitor SAHA, which was previously shown to normalize metabolic status and examined the effects of the conditioned media. We observed significant suppression of inflammatory polarization associated with decreased T-cell proliferation and recovery of mitochondrial energy metabolism.

Conclusion: This study demonstrates how the pulmonary fibroblast-derived microenvironment can activate and differentiate T-cells to trigger local inflammation, which is part of the vascular wall remodeling process in PH.

Keywords: HDAC inhibitors; T-cells; Tregs; pulmonary fibroblasts; pulmonary hypertension; γδ T-cells.

Figure 1

Single-cell RNA sequencing analysis of in vivo calf T-cells derived from pulmonary arteries of control/PH calves. (A) Analysis of mRNA levels of T cell pan (CD3E and ZAP70) and specific (CD4 for helper T-cells and CD8 for cytotoxic T-cells) marker genes in the T cell subclusters from Control (CO) and PH Distal Pulmonary Arteries (DPAs). (B) Analysis of the mRNA levels of regulatory T-cells or Treg marker gene (FOXP3) in the T cell subclusters in CO and PH DPAs. (C) Analysis of mRNA levels of cytokines in the T cell subclusters in CO and PH DPAs. Notice that if there is zero value in one of the comparison groups (CO or PH), no statistics can be computed.

Figure 2

Analysis of cytokines and markers and their mRNA expression profile of bovine/human T-cells exposed to CM-(h)CO/PH Fibs. (A) Frequency of IFNγ positive bovine/human T-cells per CD4+ T-cells (left panel) and mRNA expression of IFNγ in the period of 0-48 hrs (bovine, middle panel) and early and late time period (human, right panel) in bovine/human T-cells exposed to CM-(h)CO/PH Fibs and RPMI as a control media for early/late time period, n=3-5; Pearson correlation p=0.0132, n=3-5. (B) Frequency of IL10-positive bovine/human T-cells per CD4+ T-cells (left panel) and mRNA expression of IL10 in the period of 0-48 hrs (bovine, middle panel) and early and late time period (human, right panel) in bovine/human T-cells exposed to CM-(h)CO/PH Fibs and RPMI as a control media for early/late time period, n=3-5; Pearson correlation p=0.0476, n=3-5. (C) Frequency of TGFβ- positive bovine/human T-cells per CD4+ T-cells (left panel) and mRNA expression of TGFβ in the period of 0-48 hrs (bovine, middle panel) and early and late time period (human, right panel) in bovine/human T-cells exposed to CM-(h)CO/PH Fibs and RPMI as a control media for early/late time period, n=3-5; Pearson correlation p=0.0769, n=3-5. (D) Frequency of IL4 positive bovine/human T-cells per CD4+ T-cells (left panel) and mRNA expression of IL4 in the period of 0-48 hrs (bovine, middle panel) and early and late time period (human, right panel) in bovine/human T-cells exposed to CM-(h)CO/PH Fibs and RPMI as a control media for early/late time period, n=3-5; Pearson correlation p=0.0232, n=3-5. In mRNA expression experiments with bovine T-cells proinflammatory control was performed by LPS/nigericin treatment for 6 hrs, n=3 (orange square). p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****), not significant (ns).

Figure 3

Analysis of cytokines and T-cell markers within the FOXP3 human/bovine T-cells exposed to CM-(h)CO/PH Fibs. (A) Frequency of FOXP3 cells per bovine/human CD4+ T-cells exposed to CM-(h)CO/PH Fibs and RPMI as a control media for early/late time point, n=3-5. (B) Frequency of IL10-positive cells per FOXP3 bovine/human T-cell population exposed to CM-(h)CO/PH Fibs for early/late time point, n=3-5. (C) Frequency of TGFβ-positive cells per FOXP3 bovine/human T-cell population exposed to CM-(h)CO/PH Fibs for early/late time point, n=3-5. (D) Frequency of IL4-positive cells per FOXP3 bovine/human T-cell population exposed to CM-(h)CO/PH Fibs for early/late time point, n=3-5. p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****), not significant (ns).

Figure 4

Energy metabolism, redox status and proliferation of bovine T-cells exposed to CM-CO/PH Fibs. (A) P-PDH/PDH protein ratio of CD4+ T-cells in CM-CO/PH Fibs after 24-hour cultivation, n=4. MW of (P)PDH signal is 43kDa. (B) Lactate production expressed as media acidification (ECAR) of total (CD3+)/CD4+ T-cells in CM-CO/PH Fibs after 24-hour cultivation, n=3-5. (C) Oxidative phosphorylation activity expressed as endogenous/oligomycin-sensitive respiratory ratio of total (CD3+)/CD4+ T-cells in CM-CO/PH Fibs after 24-hour cultivation, n=3-5. (D) Maximum mitochondrial respiration analysis expressed as uncoupled/oligomycin-sensitive respiration ratio of total (CD3+)/CD4+ T-cells in CM-CO/PH Fibs after 24-hour cultivation, n=3-5. (E) Quantification of cytosolic ATP of T-cells in CM-CO/PH Fibs at early/late point of cultivation, n=4-5. (F) Proliferation of T-cells in CM-CO/PH Fibs at early/late point of cultivation, n=5. (G) Cytosolic redox status expressed as glucose-induced increase in DCF fluorescence in T-cells in CM-CO/PH Fibs after 24-hour cultivation, n=5. p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****), not significant (ns).

Figure 5

Analysis of cytokines and T-cell markers within bovine γδ+T-cell population exposed to CM-CO/PH Fibs. (A) Frequency of γδ+ cells per bovine T-cell population exposed to CM-CO/PH Fibs and RPMI as a control media at early/late point of cultivation, n=5. (B) Frequency of IL10-positive cells per γδ+ bovine T-cell population exposed to CM-CO/PH Fibs at early/late point of cultivation, n=5. (C) Frequency of TGFβ-positive cells per γδ+ bovine T-cell population exposed to CM-CO/PH Fibs at early/late point of cultivation, n=5. (D) Frequency of IL4-positive cells per γδ+ bovine T-cell population exposed to CM-CO/PH Fibs at early/late point of cultivation, n=5. p<0.01 (**), p<0.001 (***), p<0.0001 (****), not significant (ns).

Figure 6

Analysis of cytokines and markers of bovine T-cells exposed to CM-CO/PH Fibs+SAHA. (A) Fold change of frequency of IFNγ -positive bovine CD4+ T-cells exposed to CM-CO/PH Fibs+SAHA for early/late time period to CM-CO/PH Fibs, n=5. (B) Fold change of frequency of IL4-positive bovine CD4+ T-cells exposed to CM-CO/PH Fibs+SAHA for early/late time period to CM-CO/PH Fibs, n=5. (C) Fold change of frequency of IL10-positive bovine CD4+ T-cells exposed to CM-CO/PH Fibs+SAHA for early/late time period to CM-CO/PH Fibs, n=5. (D) Fold change of frequency of TGFβ -positive bovine CD4+ T-cells exposed to CM-CO/PH Fibs+SAHA for early/late time period to CM-CO/PH Fibs, n=5. (E) Fold change of frequency of FOXP3 T-cells and IL10+/TGFβ+/IL4+ cells per FOXP3 exposed to CM-CO/PH Fibs+SAHA for early/late time point to CM-CO/PH Fibs, n=4-5. (F) Fold change of frequency of γδ+ T-cells and IL10+/TGFβ+/IL4+ cells per γδ+ exposed to CM-CO/PH Fibs+SAHA for early/late time point to CM-CO/PH Fibs, n=3-5. p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****), not significant (ns).

Figure 7

Analysis of metabolism, redox status and proliferation of bovine T-cells cultured in CM-CO/PH Fibs ± SAHA. (A) Glucose fold induced endogenous respiration, oxidative phosphorylation activity (OXPHOS activity) and maximal mitochondrial respiratory capacity of T-cells cultured in CM-CO/PH Fibs ± SAHA for 24 hrs expressed as fold change to CM-CO/PH Fibs T-cells, n=4-5. (B) Lactate production expressed as media acidification (ECAR) of T-cells in CM-CO/PH Fibs ± SAHA after 24 hrs of cultivation expressed as fold change to CM-CO/PH Fibs T-cells, n=4-5. (C) P-PDH/PDH protein ratio of T-cells in CM-CO/PH Fibs ± SAHA after 24 hrs of cultivation expressed as fold change to CM-CO/PH Fibs T-cells, n=4-5. (D) Quantification of cytosolic ATP of T-cells in CM-CO/PH Fibs ± SAHA at early/late time point of cultivation expressed as fold change to CM-CO/PH Fibs T-cells, n=5. (E) Cytosolic redox status expressed as glucose-induced increase in DCF fluorescence in T-cells in CM-CO/PH Fibs ± SAHA after 24 hrs of cultivation expressed as fold change to CM-CO/PH Fibs T-cells, n=4-5. (F) Proliferation of T-cells in CM-CO/PH Fibs +/- SAHA at early/late point of cultivation, n=5. p<0.05 (*), p<0.01 (**), p<0.001 (***), p<0.0001 (****), not significant (ns).