**6. Treatments to control inflammation in burns**

#### **6.1. Traditional clinical management**

wounds heal without the complications of chronic inflammation leading to SIRS or AIRS. This is due to the fact that excessive inflammation in the early stages of healing has been identified

Burn injuries are often characterised by debilitating hypertrophic scarring, often requiring revisionary surgery (**Figure 3**). In children, scar formation following burn injury is of particular concerns, as the growing child will be restricted by non-elastic scars, which when occurring over moving joints can become functionally restrictive [59]. This is due to an excessive synthesis and deposition of ECM alongside the reduced degradation and remodelling of tissue, leading to the dense formation of collagen in long bundles rather than the normal basket weave formation [60]. Scars are also characterised by an absence of skin appendages such as hair follicles, sweat glands and nerves, which results in functional deficiency, loss of ability to regulate body temperature and absence of sensation [61]. Due to the potentially large areas which may be affected by severe scarring following burn injury, the ability to reduce scar

The link between the increased inflammatory response and the formation of scars is well established [61, 62]. It is clear that prolonged and/or excessive inflammation in the early stages of burn injury leads to excessive fibrosis and scarring [63]. In particular, the numbers of macrophages found within a wound at specific times of the healing cascade are associated with the level of fibrosis and scar formation observed [10]. Likewise, elevated TGF-β1 signalling is directly associated with increased collagen deposition and fibrosis within the healed wound

as a causative factor in the formation of scarring and fibrosis.

**5. Chronic inflammation and burn scar formation**

**Figure 3.** Post-burn hypertrophic scar on anterior chest wall. Reproduced from [59].

formation is of critical importance.

46 Hot Topics in Burn Injuries

Although minor burns heal quickly with little intervention, more severe burns require specialised clinical care to prevent hypovolemic shock, curb inflammation whilst protecting the patient from infection [6, 12]. Fluid resuscitation is often the first step in treating the severe burns patient and research generally centres on determining the optimal fluid volume to avoid cardiac and pulmonary complication, however, the effect of the fluid resuscitation strategy may also impact the inflammatory response [6]. For example, the lactate in a once preferred resuscitation fluid actually stimulates the inflammatory response, negatively affecting the prognosis for burns patients [53]. But the addition of the soluble polysaccharide, glucan phosphate to resuscitation fluid may prove useful as it has been seen to reduce pro-inflammatory cytokine expression post burn whilst increasing resistance to infection by *Pseudomonas aeruginosa* in mice [65]. The nutritional support given to the patient may also modulate the immune system [5]. Indeed, the amounts and specific types of carbohydrates and fats consumed can greatly alter the immune status. It is important to acknowledge then the impact of the dietary changes upon the immune response and investigate how different enteral feeding strategies may impact the immune profile of the patient [5, 66]. For example, a combination of arginine and Omega-3 fatty acid supplementation could be a useful addition due to the initial findings of positive effects on wound healing rates and resistance to infection, but it is not yet known if these will translate into burns patients [67]. A recent clinical trial found that supplementing with isolated soy protein, with or without fish oil was able to decrease the inflammatory response and improve wound healing in burn patients, but it is not yet known which specific compounds in the soy protein are responsible for this action [68]. Interestingly, the topical application of fatty acids isolated from various animals and plants have also been shown to have a positive effect upon burn wound healing and dampening excessive inflammation post burn injury [69], which supports the use of dietary fats in treating burns. However, other adjunct therapies which modulate the immune response have also become the focus of much research into curbing inflammation to reduce mortality and scar formation following burns.

#### **6.2. New research for the modulation of the immune system**

A number of avenues for modulating the immune system have so far been investigated which may prove useful in the treatment of burns. These include inhibiting the activation of the immune response, preventing the recruitment and activity of immune cells, interrupting the signalling pathways involved in inflammation and enhancing the resolution of the inflammatory phase.

#### *6.2.1. Inhibition of the activation of the immune response*

Curbing inflammation in burns may be achieved at the outset by inhibiting complement activation. Treatment with a C1 inhibitor in a porcine model of burn injury was found to attenuate inflammatory tissue destruction post burn [70, 71] and reduction of C4 in knockout mice prevented hypertrophic scarring following burn injury [72]. Alternatively, blocking immune cell activation using antibodies to block cytokines such as TNFα and IL-1β have been shown to be effective in vitro [52] and local reduction of TNFα using a hyaluronic acid conjugated TNFα antibody reduced necrotic burn progression in a rat model [73]. It has also been suggested that developing therapeutics to prevent NF-κB activation with antioxidants or an agent to block intracellular processes involved in its activation may be a better approach, and there is a current search for a highly specific compound [52]. Stabilisation of mast cells using cromolyn solution prevents both the rise in levels of plasma histamine and xanthine oxidase following thermal injury in rats preventing the initiation of the immune cascade and reducing the severity of the burn [34]. Treatment with pentoxifylline (PTX) immediately following burn injury in mice was found to reduce intestinal permeability and lung injury by reducing levels of intestinal IL-6 and limiting the increase in pro-inflammatory cytokine levels and inflammatory cell activation which may be useful in the prevention of SIRS [53]. Rather than preventing the activation of the immune cells though, one may also aim to reduce their numbers within the wound site.

action of a protein which acts upon the signalling pathway may prove beneficial. The protein Flightless I (Flii) has been shown to modulate TGF-β1 signalling in fibroblasts, with reduced TGF-β1 expression and collagen production in cells with reduced Flii expression [79]. Flii also directly affects the immune response, altering macrophage activation and cytokine production in vitro and increasing Flii expression in diabetic wounds is associated with increased NF-κB production and a prolonged inflammatory phase is observed in the healing of incisional wounds of mice [79–81]. The application of a Flii neutralising antibody in an animal model of the inflammatory skin condition, psoriasis, reduces pro-inflammatory cytokine expression and immune cell infiltration [82]. By treating mouse burn wounds with this antibody and reducing the expression of Flii, it was possible to decrease TGF-β1 levels and cause faster wound healing with less

The Role of the Inflammatory Response in Burn Injury http://dx.doi.org/10.5772/intechopen.71330 49

**Figure 4.** Flii neutralising antibody (FliAb) improves burn injury repair. Representative partial-thickness burn wounds treated with intradermal injection of (a) control IgG (50 μg mL−1) or (b) FliAb (50 μg mL−1) 14 days post-treatment. Dotted line indicates residual burn wound. (c) Graph showing surface wound area of partial-thickness burns treated with either IgG or FliAb post-burn injury. Representative images of (d) IgG and (e) FliAb-treated partial-thickness burns at day 14 post-injury stained with Masson's Trichrome. (f) Graph showing semi-qualitative assessment of total collagen levels within wound of IgG or FliAb-treated burn wounds. (g) Graph showing TGF-β1 fluorescence intensity after 14 days in burn wounds treated with IgG or FliAb post-burn injury. (h) Graph showing TGF-β3 fluorescence intensity after 14 days in burn wounds treated with IgG or FliAb post-burn injury. \*Denotes significance P <0.05. Results represent mean ± SEM

scar formation in mice [83] (**Figure 4**).

(n = 8). Reproduced from [83].

#### *6.2.2. Prevention of immune cell recruitment and activity*

Early studies in mice to deplete numbers of specific immune cells have shown that whilst no one immune cell is critical to wound healing, altering the recruitment or activity of these cells, provided no overt infection pre-exists, may be beneficial to the repair process and indeed may lessen the scarring observed [11]. Studies which specifically depleted macrophage numbers prior to wounding have shown that although wound closure is delayed, the wounds heal with reduced fibrosis and scar formation [74, 75]. Indeed, the targeted depletion of macrophages from the early inflammatory stage (days 0–5) of healing results in minimised scarring. Although these mice showed a reduced rate of epithelialisation during the inflammatory stage, once the mice were allowed to produce new macrophages, wound closure was rapid compared to control animals and with greatly reduced scarring [76]. It has been suggested that reducing the ability of immune cells to receive chemotactic signals may also be useful in modulating the immune response to facilitate better healing [11]. Blocking the activity of chymase released by mast cells using a specific chymase inhibitor TY51469 was effective in supressing neutrophil accumulation, reducing TGF-β1 expression and the extent of pulmonary fibrosis in mice [77] and may be a candidate therapeutic for reducing fibrosis and scarring following severe burns. Indeed, mice which lack mouse mast cell protease (mMCP)-4, the functional equivalent to human chymase, showed a much reduced level of injury following a second-degree scald burn [78].

#### *6.2.3. Interruption of immune signalling pathways*

Pharmacological agents which themselves reduce TGF-β1 signalling may also prove useful for reducing fibrosis following burn injury [11]. Alternatively, applying an antibody to interrupt the action of a protein which acts upon the signalling pathway may prove beneficial. The protein Flightless I (Flii) has been shown to modulate TGF-β1 signalling in fibroblasts, with reduced TGF-β1 expression and collagen production in cells with reduced Flii expression [79]. Flii also directly affects the immune response, altering macrophage activation and cytokine production in vitro and increasing Flii expression in diabetic wounds is associated with increased NF-κB production and a prolonged inflammatory phase is observed in the healing of incisional wounds of mice [79–81]. The application of a Flii neutralising antibody in an animal model of the inflammatory skin condition, psoriasis, reduces pro-inflammatory cytokine expression and immune cell infiltration [82]. By treating mouse burn wounds with this antibody and reducing the expression of Flii, it was possible to decrease TGF-β1 levels and cause faster wound healing with less scar formation in mice [83] (**Figure 4**).

*6.2.1. Inhibition of the activation of the immune response*

48 Hot Topics in Burn Injuries

*6.2.2. Prevention of immune cell recruitment and activity*

*6.2.3. Interruption of immune signalling pathways*

Curbing inflammation in burns may be achieved at the outset by inhibiting complement activation. Treatment with a C1 inhibitor in a porcine model of burn injury was found to attenuate inflammatory tissue destruction post burn [70, 71] and reduction of C4 in knockout mice prevented hypertrophic scarring following burn injury [72]. Alternatively, blocking immune cell activation using antibodies to block cytokines such as TNFα and IL-1β have been shown to be effective in vitro [52] and local reduction of TNFα using a hyaluronic acid conjugated TNFα antibody reduced necrotic burn progression in a rat model [73]. It has also been suggested that developing therapeutics to prevent NF-κB activation with antioxidants or an agent to block intracellular processes involved in its activation may be a better approach, and there is a current search for a highly specific compound [52]. Stabilisation of mast cells using cromolyn solution prevents both the rise in levels of plasma histamine and xanthine oxidase following thermal injury in rats preventing the initiation of the immune cascade and reducing the severity of the burn [34]. Treatment with pentoxifylline (PTX) immediately following burn injury in mice was found to reduce intestinal permeability and lung injury by reducing levels of intestinal IL-6 and limiting the increase in pro-inflammatory cytokine levels and inflammatory cell activation which may be useful in the prevention of SIRS [53]. Rather than preventing the activation of the immune cells though, one may also aim to reduce their numbers within the wound site.

Early studies in mice to deplete numbers of specific immune cells have shown that whilst no one immune cell is critical to wound healing, altering the recruitment or activity of these cells, provided no overt infection pre-exists, may be beneficial to the repair process and indeed may lessen the scarring observed [11]. Studies which specifically depleted macrophage numbers prior to wounding have shown that although wound closure is delayed, the wounds heal with reduced fibrosis and scar formation [74, 75]. Indeed, the targeted depletion of macrophages from the early inflammatory stage (days 0–5) of healing results in minimised scarring. Although these mice showed a reduced rate of epithelialisation during the inflammatory stage, once the mice were allowed to produce new macrophages, wound closure was rapid compared to control animals and with greatly reduced scarring [76]. It has been suggested that reducing the ability of immune cells to receive chemotactic signals may also be useful in modulating the immune response to facilitate better healing [11]. Blocking the activity of chymase released by mast cells using a specific chymase inhibitor TY51469 was effective in supressing neutrophil accumulation, reducing TGF-β1 expression and the extent of pulmonary fibrosis in mice [77] and may be a candidate therapeutic for reducing fibrosis and scarring following severe burns. Indeed, mice which lack mouse mast cell protease (mMCP)-4, the functional equivalent to human chymase,

showed a much reduced level of injury following a second-degree scald burn [78].

Pharmacological agents which themselves reduce TGF-β1 signalling may also prove useful for reducing fibrosis following burn injury [11]. Alternatively, applying an antibody to interrupt the

**Figure 4.** Flii neutralising antibody (FliAb) improves burn injury repair. Representative partial-thickness burn wounds treated with intradermal injection of (a) control IgG (50 μg mL−1) or (b) FliAb (50 μg mL−1) 14 days post-treatment. Dotted line indicates residual burn wound. (c) Graph showing surface wound area of partial-thickness burns treated with either IgG or FliAb post-burn injury. Representative images of (d) IgG and (e) FliAb-treated partial-thickness burns at day 14 post-injury stained with Masson's Trichrome. (f) Graph showing semi-qualitative assessment of total collagen levels within wound of IgG or FliAb-treated burn wounds. (g) Graph showing TGF-β1 fluorescence intensity after 14 days in burn wounds treated with IgG or FliAb post-burn injury. (h) Graph showing TGF-β3 fluorescence intensity after 14 days in burn wounds treated with IgG or FliAb post-burn injury. \*Denotes significance P <0.05. Results represent mean ± SEM (n = 8). Reproduced from [83].

The potential therapy has been further investigated in a model of hypertrophic scarring, where it was found that application of Flii antibodies resulted in less fibrosis by reducing the number of myofibroblasts within the wound [84]. Further investigation into the use of neutralising antibodies to dampen inflammation and reduce fibrosis in human burns patients is clearly warranted.

**Author details**

South Australia, Australia

1953;**40**(164):588-596

**110**(9):3513-3518

**References**

364-373

**2017**:17

2015;**23**(1):8-12, 4-5

Xanthe L. Strudwick and Allison J. Cowin\*

\*Address all correspondence to: allison.cowin@unisa.edu.au

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Regenerative Medicine, Future Industries Institute, University of South Australia, Adelaide,

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#### *6.2.4. Enhancement of the resolution of the inflammatory response*

New therapies are also being developed to resolve post burn inflammation quickly and lead to better healing outcomes. Lipid mediators, such as the resolvins and lipoxins, which stimulate the resolution of the inflammatory phase, have become of particular interest in recent years [11]. Resolvin D2 has recently been found to restore burn neutrophil directionality and can reduce neutrophil numbers and minimise secondary necrosis in burns [85, 86]. The use of phototherapy has been shown to significantly reduce the number of immune cells within a rat burn wound, increasing angiogenesis and collagen deposition [87] and may herald a new area of research for the development of devices to treat burn wounds. Novel biomaterials are also being investigated in the treatment of burn injuries, not only to provide a provisional matrix or augment skin grafting, but are also being assessed for their ability to modulate the immune response [88]. Fibrin-based hydrogels delivered into pig burn wounds prevented contracture but also reduced immune cells within the hydrogel as well as reducing neutrophil and macrophage numbers within the in the surrounding granulation tissue on day 7 post burn [89]. It is expected that future therapies will aim to provide the dual roles of enhancing healing outcomes whilst preventing the excessive systemic inflammatory effects post burn.
