Immunology of Wound Healing

Danielle Tartar, MD, PhD Assistant Clinical Professor Interim Director of Inpatient Dermatology University of California - Davis Outline

• Traditional model of wound healing • Role of the immune system on Inflammatory (1-3 days), Proliferative (4-21 days), Remodeling (21 days – 1 year) phases of wound healing . Signals that help transition from Inflammatory – Proliferative phase Wound Healing • Chronic wounds present a significant problem to patients and the healthcare system – At risk: diabetes, immunocompromised, elderly, impaired arterial/ venous circulation • Chronic wounds affect >6 million people in the US • Cost of over $25 billion annually • With current standard of care, only 30% of patients heal • A greater understanding of the events involved in wound healing is required

Powers et al. Wound healing and treating wounds. JAAD CME April 2016 Fahs et al. New insights into MicroRNAs in skin wound healing. IUBMB Life 2015 Otero-Vinas et al. Mesenchymal stem cells for chronic wounds: the spectrum from basic to advanced therapy. Adv Wound Care 2016 Wound Healing • Complex over-lapping but distinct series of events – Recruitment of immune cells – Phagocytosis of debris and prevention of infection – Generation of new matrix and vasculature – Re-epithelialization of keratinocytes – Remodeling of wounds

Deregulation of any step results in impaired healing

Traditional Model of Wound Healing

Hemostasis • Platelet plug • Danger signals Remodeling • Scar

Inflammation • Phagocytosis of debris Proliferation • Prevention of infection • ECM • Angiogenesis X • Re-eptithelialization

Portou, et al. The innate immune system, toll-like receptors and dermal wound healing: a review. Vascular Pharmacology 2015 Hemostasis / Inflammatory Phase Hemostasis • Begins within minutes • Platelets aggregate, attach to exposed collagen and are activated – Platelet plug – Vasoconstriction (initial wound blanching, 10- 15min) followed by vasodilation – Activation of tissue resident cells  proinflammatory chemokines  directional migration of inflammatory cells and endothelial cells

Balaji et al. Chemokine involvement in fetal and adult wound healing. Adv Wound Care 2015 Inflammatory Phase: CXCL8 and Neutrophil Migration

• CXCL8 family chemokines – CXCL1, CXCL2 (MIP-2), CXCL3, CXCL5, CXCL6, CXCL7, CXCL8 – Bind glycosaminoglycans on cell walls and ECM to create gradients for neutrophil trafficking • Bind to CXCR1 and CXCR2 on neutrophils

De Oliveira et al. Neutrophil migration in infection and wound repair: going forward in reverse. Nature reviews immunol 2016 McDonald, B et al. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 2010. Beyond CXCL8

• Neutrophils have over 30 different R that can modulate migration

Wilgus et al. Neutrophils and wound repair: positive and negative reactions. Adv Wound Care 2013 De Oliveira et al. Neutrophil migration in infection and wound repair: going forward in reverse. Nature reviews immunol 2016 Beyond CXCL8 • First signals are released directly from necrotic/damaged cells: – Damage-associated molecular patterns (DAMPs) • DNA, proteins, ECM, ATP, uric acid) – PAMPs in the setting of infection • Bind receptors on neutrophils directly: PRR (TLR, NLR), ATP binds P2X and P2YR • Stimulate the production of gradients that drive neutrophil

migration (H2O2, CXCL8, LTB4)

Wilgus et al. Neutrophils and wound repair: positive and negative reactions. Adv Wound Care 2013 De Oliveira et al. Neutrophil migration in infection and wound repair: going forward in reverse. Nature reviews immunol 2016 NETosis Delays Wound Healing in Diabetes • NETs: • DNA structures released due to chromatin decondensation + bactericidal proteins • NETs increased in wounds and blood of patients with non-healing foot ulcers, correlated with diabetes • Inhibition of NET function improves wound healing in (diabetic) mouse model

Fadini et al. NETosis Delays Diabetic Wound Healing in Mice and Humans. Diabetes 2016. Delgado-Rizzo et al. Neutrophil Extracellular Traps and Its Implications in Inflammation: An Overview. Frontiers in Immunology 2017

Inflammatory Phase: Neutrophil Function in Wounds

• Neutrophils must be cleared from the wound to allow healing • Apoptosis / engulfment by macrophages • Reverse neutrophil migration and/or re- enter the circulation (reverse transendothelial migration (rTEM)) • Possible mechanism to locally resolve inflammation • Neutrophil reverse migration may lead to activated neutrophils being redistributed to other locations in the body, contributing to inflammation elsewhere

De Oliveira et al. Neutrophil migration in infection and wound repair: going forward in reverse. Nature reviews immunol 2016 Proliferative

Macrophages and Wound Healing • Critical to wound healing – depletion of Mφ results in delayed wound healing • Different phenotypes are involved in all phases of wound healing

Minutti et al. Tissue-specific contributions of macrophages to wound healing. Seminars in cell and developmental biology 2017 Koh et al. Inflammation and wound healing: The role of the macrophage. Expert rev mol med 2011. Mirza et al. Selective and specific macrophage ablation is detrimental to wound healing in mice. Am J. Pathol 2009 How do Mφ transition to a pro-repair phenotype? • IL-4 / IL-13 induce the alternative Mφ phenotype in vitro • Efferocytosis (phagocytosis of apoptotic cells) of neutrophils – Inhibits IL-1b, IL-8, GCSF, TNFα and increases TGFb, PGE2, PDGF by Mφ • Adenosine 2A R (A2AR), TLR signaling, IL-10, glucocorticoids, prostaglandins, and modulators of glucose and lipid metabolism • Treg cells, miRNAs, MSC, serotonin signaling • Iron overload can prevent this transition

Landen et al. Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci 2016 Nosabaum et al. Regulatory T cells facilitate cutaneous wound healing. J Immunol 2016 Pinhal -Enfield et al. An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors. Am J Pathol 2003 Regulski et al. Mesenchymal Stem Cells: “Guardians of Inflammation” Wounds 2016 Fahs et al. New insights into MicroRNAs in skin wound healing. IUBMB Life 2015 Fadok et al. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF JCI 1998 Daley et al. The phenotype of murine wound macrophages. J Leukoc Biol 2010 Koh et al. Inflammation and wound healing: The role of the macrophage. Expert rev mol med 2011. How do Mφ transition to a pro-repair phenotype? A role for serotonin signaling • MSC produce high levels of serotonin • Fluoxetine and serotonin increase re- epithelialization in vivo in a diabetic wound model • At day 10, neutrophils in the wound bed are markedly decreased and macrophages are markedly increased with both fluoxetine and serotonin treatment • Fluoxetine and serotonin influence macrophage polarization towards an anti- inflammatory / pro-repairative phenotype in vitro • Increased IL-10, TGF • Decreased TNF 𝛃𝛃 𝛼𝛼

Tartar, Nguyen, Isseroff. Manuscript under submission. Proliferative • Angiogenesis – VEGF, PDGF, bFGF, thrombin • Formation of granulation tissue – Formed during days 3-5 in healthy skin (overlaps with inflammatory phase) – Fibroblasts, granulocytes, Mφ, blood vessels, collagen bundles • Epithelialization – Keratinocyte activation and migration begins within hours after injury – Resident epidermal stem cells: • basal layer of the interfollicular epidermis • bulge region of the outer follicular root

Bolognia, 3rd ed

Landen et al. Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci 2016 Koh et al. Inflammation and wound healing: The role of the macrophage. Expert rev mol med 2011.

The Immune System and Angiogenesis

• Blood vessel networks expand in a 2-step process • Vessel sprouting (VEGF) • Vessel anastomosis • Macrophages may not only produce pro- angiogenic signals, but may act as vascular fusion chaperones • Angiogenesis is impaired in macrophage deficient models

Fantin et al. Tissue macrophages act as cellular chaperones for vascular anastomosis downstream of VEGF-mediated endothelial tip cell induction. Vascular Biology 2010.

The Immune System and ECM Formation and Re-epithelialization

EGF, TGFα Keratinocytes

Pro- Fibronectin, Repair tenascin C, KGF, EGF Mφ laminin 332 Fibronectin

PDGF, TNFα, IL-1, IL-6

Fibroblasts Fibrin, Fibronectin, Vitronectin, Platelets, Type I / III Collagen

Rodero, M et al. Skin wound healing modulation by macrophages. Int J Clin Exp Path 2010. Rouselle, P et al. Extracellular matrix contribution to skin wounds re-epithelialization. Matrix Biology 2018. Lin, Z et al. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice. JLB 2013.

IL-6 and Wound Healing • Induces expression of MIP-2 and MIP- 1α, which are chemotactic for neutrophils and monocytes/macrophages, respectively • IL-6 KO mice showed decreased neutrophils and macrophages in wounds • IL-6 induces IL-1 expression along with key adhesion molecules VCAM-1 and ICMA-1 • IL-6 induces collagen / procollagen production from fibroblasts and fat- storing cells in the liver through induction of TGF-β1 gene expression.

Lin, Z et al. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice. JLB 2013.

Remodeling

• Remodeling occurs ~1year • Scar formation is the end result of wound healing in adults • Collagen III in the ECM is replaced by collagen I (higher tensile strength but takes longer time to deposit) • Angiogenesis slows, mature avascular / acellular environment is formed • Some skin components (hair follicles, sweat glands) cannot be recovered • 80 % of the original tensile strength

Landen et al. Transition from inflammation to proliferation: a critical step during wound healing. Cell Mol Life Sci 2016 Van De Water et al. Mechanoregulation of the Myofibroblast in Wound Contraction, Scarring, and Fibrosis: Opportunities for New ∼Therapeutic Intervention. Adv Wound Care 2013 Tilkorn, S et al. Skin Wound Healing: An Update on the Current Knowledge and Concepts. Eur Surg Res 2017 Remodeling: Myofibroblasts and Wound Contraction • Mechanical tension, TGFβ, EDA fibronectin dermal fibroblasts differentiate into myofibroblasts – Express α-smooth muscle actin (SMA) and contract the wound – Secrete MMP, TIMPs and contribute to scar remodeling

Beyond Contraction: Myofibroblasts and Fat Regeneration? • Dogma: wound healing in adults results in scar with excess collagen / absence of hair follicles and cutaneous fat • Recent data shows that hair follicles can regenerate in mouse model of wounding (neogenic hair follicles) • Myofibroblasts can differentiate into cutaneous fat, restoring adipose tissue in scars (myofibrobasts previously thought to be terminally differentiated!) – Requires neogenic hair follicles, which trigger BMP signaling and activation of adipocyte transcription factors

Plikus, M et al. Regeneration of fat cells from myofibroblasts during wound healing. Science 2017 Fig. 1 New adipocytes only regenerate around new hair follicles during wound healing

Plikus, M et al. Regeneration of fat cells from myofibroblasts during wound healing. Science 2017 Beyond Contraction: Myofibroblasts and Fat Regeneration?

• Myofibroblast lineage reprogramming to adipocytes may represent a target for influencing regeneration rather than scarring of tissue in the future

Plikus, M et al. Regeneration of fat cells from myofibroblasts during wound healing. Science 2017 Remodeling – Formation of Keloids / Hypertrophic Scars

Hypertrophic Scar Keloid

Sites Shoulders, neck, Anterior chest, presternum, knees, shoulders, earlobe, ankles upper arms, cheeks Time course Begins within 4-8 Can occur years later weeks of wound Appearance Does not extend Projects beyond beyond initial injury original wound margins Histology Type III collagen Disorganized type I bundles parallel to and III collagen. Poor epidermis. Acidic vascularization with mucopolysaccharide scattered, dilated blood vessels

Gauglitz et al. Hypertrophic Scarring and Keloids: Pathomechanisms and Current and Emerging Treatment Strategies. Mol Med 2011

The Role of the Immune System in Keloid / Hypertrophic Scar Formation • Normally, ECM is degraded and type III collagen is replaced by type I • Delicate balance between ECM protein degradation and deposition • CD4 T cell phenotypes help regulate this balance – Th1 (IFNγ, IL-12) attenuate tissue fibrosis – Th2 (IL-4, IL-5, IL-13) strongly linked to fibrogenesis • Not only immune-mediated (keloid fibroblasts have increased growth factor receptors)

Gauglitz et al. Hypertrophic Scarring and Keloids: Pathomechanisms and Current and Emerging Treatment Strategies Mol Med 2011 Wynn et al. Fibrotic diseases and the Th1/Th2 paradigm. Nature Reviews Immunol 2004.

Wound Healing • Complex over-lapping but distinct series of events coordinated in part by immune cells and inflammatory mediators – Recruitment of immune cells • Neutrophils (DAMP, IL-8) • Monocytes -> Macrophages – Phagocytosis of debris and prevention of infection • Transition from pro- anti- inflammatory Macrophages – Generation of new matrix and vasculature, re- epithelialization of keratinocytes – Formation of scar, remodeling

Acknowledgements

• Dr. Sam Hwang • Dr. R. Rivkah Isseroff • Chuong Nguyen, PhD candidate and Isseroff laboratory, UC Davis