Abstract
BACKGROUND Lymphedema is a global health problem with no effective drug treatment. Enhanced T cell immunity and abnormal lymphatic endothelial cell (LEC) signaling are promising therapeutic targets for this condition. Sphingosine-1-phosphate (S1P) mediates a key signaling pathway required for normal LEC function, and altered S1P signaling in LECs could lead to lymphatic disease and pathogenic T cell activation. Characterizing this biology is relevant for developing much-needed therapies.
METHODS Human and mouse lymphedema was studied. Lymphedema was induced in mice by surgically ligating the tail lymphatics. Lymphedematous dermal tissue was assessed for S1P signaling. To verify the role of altered S1P signaling effects in lymphatic cells, LEC-specific S1pr1-deficient (S1pr1LECKO) mice were generated. Disease progression was quantified by tail-volumetric and -histopathological measurements over time. LECs from mice and humans, with S1P signaling inhibition, were then co-cultured with CD4 T cells, followed by an analysis of CD4 T cell activation and pathway signaling. Finally, animals were treated with a monoclonal antibody specific to P-selectin to assess its efficacy in reducing lymphedema and T cell activation.
RESULTS Human and experimental lymphedema tissues exhibited decreased LEC S1P signaling through S1PR1. LEC S1pr1 loss-of-function exacerbated lymphatic vascular insufficiency, tail swelling, and increased CD4 T cell infiltration in mouse lymphedema. LECs, isolated from S1pr1LECKO mice and co-cultured with CD4 T cells, resulted in augmented lymphocyte differentiation. Inhibiting S1PR1 signaling in human dermal LECs (HDLECs) promoted T helper type 1 and 2 (Th1 and Th2) cell differentiation through direct cell contact with lymphocytes. HDLECs with dampened S1P signaling exhibited enhanced P-selectin, an important cell adhesion molecule expressed on activated vascular cells. In vitro, P-selectin blockade reduced the activation and differentiation of Th cells co-cultured with shS1PR1-treated HDLECs. P-selectin-directed antibody treatment improved tail swelling and reduced Th1/Th2 immune responses in mouse lymphedema
CONCLUSION This study suggests that reduction of the LEC S1P signaling aggravates lymphedema by enhancing LEC adhesion and amplifying pathogenic CD4 T cell responses. P-selectin inhibitors are suggested as a possible treatment for this pervasive condition.
What is New?
Lymphatic-specific S1pr1 deletion exacerbates lymphatic vessel malfunction and Th1/Th2 immune responses during lymphedema pathogenesis.
S1pr1-deficient LECs directly induce Th1/Th2 cell differentiation and decrease anti-inflammatory Treg populations.
Peripheral dermal LECs affect CD4 T cell immune responses through direct cell contact.
LEC P-selectin, regulated by S1PR1 signaling, affects CD4 T cell activation and differentiation.
P-selectin blockade improves lymphedema tail swelling and decreases Th1/Th2 population in the diseased skin.
What Are the Clinical Implications?
S1P/S1PR1 signaling in LECs regulates inflammation in lymphedema tissue.
S1PR1 expression levels on LECs may be a useful biomarker for assessing predisposition to lymphatic disease, such as at-risk women undergoing mastectomy
P-selectin Inhibitors may be effective for certain forms of lymphedema
Competing Interest Statement
The authors have declared no competing interest.
Funding Statement
This work was supported by National Institutes of Health grants HL141105-4 (to MRN) and HL150583-02 (to XJ)
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All human tissues were approved by the Stanford University Institutional Review Board (protocol 7781). Adult patients with primary and acquired lymphedema were assessed in this study. Control samples were obtained from the healthy contralateral limb of the same patient. All animal studies were approved by the VA Palo Alto Institutional Animal Care and Use Committee (IACUC). Human buffy coats were purchased from Stanford Blood Center.
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