E. H. Steen1, X. Wang1, S. Balaji1, H. Li1, M. Fahrenholtz1, M. Butte4, P. Bollyky3, S. Keswani1,2 1Baylor College Of Medicine,Department Of Surgery,Houston, TEXAS, USA 2Texas Children’s Hospital,Department Of Pediatric Surgery,Houston, TEXAS, USA 3Stanford University,Department Of Medicine, Division Of Infectious Diseases And Geographic Medicine,Palo Alto, CALIFORNIA, USA 4University Of California, Los Angeles,Department Of Pediatrics, Division Of Allergy And Immunology,Los Angeles, CALIFORNIA, USA
Introduction: Lymphocytes, specifically T cells, have been shown to have a salutary effect in tissue repair by producing an anti-inflammatory milieu, which can result in decreased fibrosis. Further, it is established that tension impairs wound repair and increases fibrosis. How exactly the mechanical environment of a wound is sensed and transduced and how it affects the behavior of immune cells has not been fully elucidated. We hypothesize that select populations of lymphocytes are mechanosensitive and influence fibrosis and the dermal response to tissue injury.
Methods: In a loss-of-function experiment, excisional stented wounds were made in mice devoid of T and B cells (SCID) and compared to wild type (WT) mice for degree of inflammation (CD45+ lymphocytes and F4/80+ macrophages) and fibrosis (trichrome, α -SMA staining). Adoptive transfer experiments were performed on SCID mice by adding the following lymphocyte populations from WT mice: 1) total lymphocytes (TL); 2) CD4+CD25-T cells; 3) CD4+CD25+T cells. Cutaneous wounds were then created in the syngeneic mice. Wound samples were harvested and analyzed as above. To understand the role of mechanosensitivity, healthy human donor lymphocytes were embedded in low, medium and high density hydrogels to mimic extracellular matrix (ECM) stiffening as would occur in fibrosis; subsequent gene expression was assessed by PCR array. This experiment was repeated with Pyk2 inhibitor to block lymphocyte mechanosensation. Data presented as means; p-values by ANOVA and t-test.
Results: SCID mice demonstrate increased dermal fibrosis (increased trichrome staining on days 14 and 30; α -SMA expressed on IHC on day 7) and an exaggerated inflammatory infiltrate compared to WT mice (CD45: SCID 34.5% vs WT 21%; F4/80: SCID 29.4% vs WT 3.2%; p>0.05). Adoptive transfer of all three T cell types was successful and reduces inflammatory infiltrates as compared to control SCID mice (neutrophils: TL 12.9%; C4+CD25-T cells 16.5%; CD4+CD25+T cells 1%; macrophages: TL 5.8%; CD4+CD25-T cells 25%; CD4+CD25+T cells 22.2%). Fibrosis decreases in the TL and CD4+CD25+ subpopulations (decreased trichrome staining on day 7). On PCR array of 84 genes, expression of CXCL5, TLR7, C3, and CCR2 in human lymphocytes changes in response to increasing ECM stiffness (CXLC5 323-fold, TLR7 7.4-fold, C3 20-fold, and CCR2 4.5-fold higher in high vs low density gels). The addition of Pyk2 inhibitor to the system returned gene expression to control values.
Conclusion: Our data suggests that lymphocytes are mechanosensitive and respond to increasing tension by enhancing expression of inflammatory cytokines and chemokines, in part via a Pyk2-dependent mechanism. It also suggests that certain T cell subsets have a significant role in governing the dermal fibrotic phenotype. Investigating the molecular cues between mechanical forces and lymphocytes in dermal scarring may lead to the identification of anti-fibrotic agents for the skin and beyond.