The Gas6/Axl pathway regulates many cell functions and is implicated in hypertension. In this study we aimed to investigate the role of Axl in immune cells on initiation and progression of salt-dependent hypertension. Deoxycorticosterone-acetate (DOCA; 75mg/60days release) and salt hypertension was induced for 1week or 6weeks in Axl chimeras generated by bone marrow (BM) transplant to restrict Axl deficiency to hematopoietic or non-hematopoietic compartments. Depletion of Axl in hematopoietic cells (Axl−/− →Axl+/+) reduced (133±2mmHg) increase in systolic blood pressure (BP) compared to other Axl chimeras (~150mmHg) 1week after DOCA-salt. Urine protein and renal oxidative stress were lowest in Axl−/− →Axl+/+ at 1week after DOCA-salt. Compensatory increase in Gas6 in kidneys of recipient Axl−/− may affect kidney function and BP in early phase of hypertension. Flow cytometry on kidneys from Axl−/− →Axl+/+ showed increase in total leukocytes, B and dendritic cells and decrease in macrophages compared to Axl+/+ →Axl+/+. These immune changes were associated with decrease in pro-inflammatory gene expression, in particular interferon gamma. Systolic BP returned to baseline in Axl−/− →Axl+/+ and Axl−/− →Axl−/− but remained increased in Axl+/+ →Axl+/+ and Axl+/+ →Axl−/− chimeras after 6weeks of DOCA-salt. Vascular apoptosis was increased in the global Axl−/− chimeras in the late phase of hypertension. In summary, we found that expression of Axl in hematopoietic cells is critical for kidney pathology in early phase of salt-dependent hypertension. However, Axl in both hematopoietic and non-hematopoietic lineages contributes to the late phase of hypertension.

Objective Survival of immune and non-immune cells relies on Axl, a receptor tyrosine kinase, which is implicated in hypertension. Activated T lymphocytes are involved in regulation of high blood pressure. The goal of the study was to investigate the role of Axl in T lymphocyte functions and its contribution to salt-dependent hypertension. Approach and Results For the first time we report increased apoptosis in peripheral blood from Axl−/− mice due to lower numbers of white blood cells mostly lymphocytes. In vitro studies showed modest reduction in interferon gamma production in Axl−/− Th1 cells. Axl did not affect basic proliferation capacity or production of interleukin 4 in Axl−/− Th2 cells. However, competitive repopulation of Axl−/− bone marrow or adoptive transfer of Axl−/− CD4+ T cells to Rag1−/− mice showed robust effect of Axl on T lymphocytes expansion in vivo. Adoptive transfer of Axl−/− CD4+ T cells was protective in a later phase of deoxycorticosterone-acetate and salt hypertension. Reduced numbers of CD4+ T cells in circulation and in perivascular adventitia decreased vascular remodeling and increased vascular apoptosis in the late phase of hypertension. Conclusions These findings suggest that Axl is critical for survival of T lymphocytes especially during vascular remodeling in hypertension.

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Introduction: Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl. Methods and Results: We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p<0.05) in Axl-/- (396±23) compared to Axl+/+ (512±42) under Th1-priming. However, Axl had no effect on interleukin 4 (IL-4, ng/mL) production under Th2 polarizing conditions. Intracellular staining of the Th1/Th2 cells with IFN-γ and IL-4 antibodies by flow cytometry confirmed expression of cytokines in culture media. Complete blood counts showed that Axl-/- mice had significantly lower white blood cells due to decreased numbers of lymphocytes (4.5±0.7x10 9 ) compared to Axl+/+ mice (7.8±0.7x10 9 ). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice. Conclusions: Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.

Introduction: Axl, a receptor tyrosine kinase, is required for vascular and immune cell survival. We sought to investigate the effects of Axl on T lymphocyte survival during deoxycorticosterone acetate (DOCA)-salt hypertension in mice. Methods and Results: We found significant reduction in systolic blood pressure (BP) after 5-6 weeks of DOCA-salt in RAG1-/- mice after adoptive transfer of CD4+ T cells from Axl knockout (Axl-/- →RAG1-/-) compared to transferred CD4+ T cells from wild type (Axl+/+ →RAG1-/-) mice. Media area of the mesenteric artery was significantly lower in Axl-/- →RAG1-/- (4.2±0.7x10 3 m 2 ) vs. Axl+/+ →RAG1-/- (6.0±0.9x10 3 m 2 ) or Axl+/+ (6.8±0.6x10 3 m 2 ) mice. There was significant decrease in interferon gamma production by the T cells from Axl-/- (396±23 ng/mL) compared to Axl+/+ (512±42 ng/mL) after T h 1-priming. The number of carboxyfluorescein succinimidyl ester-positive cells in 6 th division was dramatically declined in Axl-/- (~0.3%) vs. Axl+/+ (~1.8%) in culture. Accordingly, we found lower number of lymphocytes in blood from Axl-/- (4.5±0.7x10 9 ) compared to Axl+/+ (7.8±0.7x10 9 ) mice. Blood leukocyte apoptosis was 2.5-fold higher in Axl-/- mice. We next investigated repopulation capacities of the hematopoietic cells from Axl-/- vs. Axl+/+ mice. There was significant decrease in Axl-/- CD3+ T cells (21±3 %) than Axl+/+ (49±3 %) in spleen after 8 weeks of competitive repopulation of bone marrow-derived cells. However, we found even greater reduction of Axl-/- T lymphocytes (15±1 %) vs. Axl+/+ T lymphocytes (52±6 %) in peripheral blood after 8 weeks of competitive repopulation. Finally, percentage of apoptotic cells was the greatest in the media (20±7 %) and adventitia (13±5 %) from Axl-/- →RAG1-/- mice compared to vascular apoptosis (6-14 % in media; and 6-9 % in adventitia) in other groups after 6 weeks of DOCA-salt. Conclusions: Our data suggest that Axl-dependent survival of the T lymphocytes is crucial for the late increase in BP in DOCA-salt hypertension.