Cumulative experimental and clinical evidence has demonstrated that cryopreserved human amniotic membrane possesses potent anti-inflammatory, anti-scarring and anti-angiogenic actions. In the past several years, our research studies have revealed new insights about the molecular candidate responsible for these actions. In this review, we will only focus on AM’s anti-inflammatory action and its therapeutic implication.

HC-HA/PTX3 Complex in AM

In 2006, we first reported that AM stromal matrix exerts an anti-inflammatory action by inducing apoptosis of IFN-γ–activated monocyte/macrophage RAW264.7 cells (See Figure 1) and that such an action is not caused by nitric oxide but instead by the downregulation of anti-apoptotic NF-κB and Akt-FKHR signaling pathways.1 Subsequently in 2008, we demonstrated that such an anti-inflammatory action is retained in soluble AM extract (AME). AME upregulates IL-10, downregulates TNF-α and IL-6, and suppresses the activation of RAW264.7 cells by IFN-γ, LPS and IFN-γ/LPS.2 These findings suggest the key molecule(s) in AM responsible for its anti-inflammatory and other biological actions can be extracted and possibly be identified and isolated.

Indeed, in 2009, we successfully purified a complex designated as “HC-HA/PTX3” from AME.3 HC-HA/PTX3, first found in cumulus-oocyte complex (COC) surrounding the ovulated oocyte, is vital for subsequent fertilization.4,5 It is formed by tight association between pentraxin 3 (PTX3) and HC-HA, which consists of high molecular weight hyaluronic acid covalently linked to heavy chain 1 (HC1) of inter-α-trypsin inhibitor. IαI is mainly secreted by the liver and present in the blood at considerably high concentrations (0.15 to 0.5 mg/ml).6 It is composed of a common light chain and two heavy chains (HC1 and HC2). The light chain is a typical proteoglycan molecule having a single chondroitin four-sulfate chain.
 
Figure 1. Amniotic membrane induces apoptosis in IFN-γ-activated RAW264.7 cells measured by LIVE/DEAD staining.1
Its core protein, known as bikunin, contains two tandem Kunitz-type protease inhibitory domains that contribute to the protease inhibitory activity. In IαI, a bikunin molecule is linked to HC1 and HC2 via a unique ester bond.6,7 IαI is usually only found in serum and ingresses into tissue spaces as a consequence of increased vascular permeability at sites of inflammation. Because AM is an avascular tissue, we then investigated how AM cells can produce HC-HA/PTX3 without having a direct access to serum IαI. In 2012, our studies unraveled that human amniotic epithelial and stromal cells constitutively express the individual HCs (e.g., HC1 and HC2) as well as the bikunin, therefore producing IαI in AM locally.8 Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronic acid-binding protein normally only induced under inflammatory conditions.9-11 It acts as a catalyst to transfer both HC1 and HC2 from IαI to HA for HC-HA complex formation. Our studies showed that human amniotic epithelial and stromal cells constitutively produce TSG-6 without requirement of stimulation by pro-inflammatory cytokines (e.g., IL-1 and TNF-α).8 In addition, PTX3, a prototypic long pentraxin that plays a critical role in innate immunity,12,13 is also an important structural component of COC. PTX3-deficient mice display a severe deficiency in female fertility due to defective assembly of the HA-rich matrix around the oocyte in the COC.5 PTX3 can be produced by mouse cumulus cells during cumulus expansion and localized within the matrix.5 It is believed that PTX3 makes multiple contacts to HCs of HC-HA complexes to provide structural integrity to the cumulus matrix,14-16 implicating a nodal activity of PTX3 depending on its multimeric organization (i.e., octamers, tetramers and dimers). Interestingly, we have found PTX3 is also constitutively expressed and secreted by AM epithelial and stromal cells as an integral component of the HC-HA/PTX3 complex.17 Collectively, for the first time our biochemical studies have shown that HC-HA/PTX3, which is originally found in COC,5 is uniquely produced and can be purified from AM.

Retains Anti-inflammatory Action

Our studies have also shown that HC-HA/PTX3 purified from AM retains AM’s anti-inflammatory action by exerting the following effects:

1) HC-HA/PTX3 promotes apoptosis of pro-inflammatory but not resting neutrophils and macrophages. During inflammation, neutrophils are among the first recruited to engulf pathogens and damaged tissues before their eventual apoptosis.18-20 Pathologically, delayed neutrophil apoptosis leads to a prolonged inflammation, which is a hallmark of many inflammatory diseases.21,22 We have reported that apoptosis of freshly-isolated neutrophils activated by fMLP or LPS is promoted only when they are treated by soluble HC-HA/PTX3, but not HA or PBS control.23 We also report that soluble HC-HA/PTX3, the same as AM and AME, dose-dependently promotes apoptosis of RAW264.7 cells activated by IFN-γ, LPS, or IFN-γ/LPS.2,3,23

2) HC-HA/PTX3 enhances phagocytosis of apoptotic neutrophils by macrophages. Clearance of apoptotic neutrophils by macrophages is essential for inflammation resolution.24-26 We have reported that soluble HC-HA/PTX3, but not HA, is effective in enhancing phagocytosis of apoptotic neutrophils by resting macrophages (about sevenfold vs. PBS control). Meanwhile, immobilized HC-HA/PTX3 is more potent in promoting phagocytosis of apoptotic neutrophils by LPS-activated macrophages (about 2.5-fold).23

3) HC-HA/PTX3 polarizes M2 macrophages. Macrophages may undergo classical M1 activation to express pro-inflammatory IL-12, which together with IL-23 activates Th1 and Th17 lymphocytes.27 Consequently, M1 polarization leads to many chronic inflammatory diseases such as arthritis, atherosclerosis and diabetes. In contrast, they may also undergo alternative M2 activation, which expresses anti-inflammatory IL-10, activates T regulatory cells (Tregs) and promotes wound healing without fibrosis.28-30 We have demonstrated that immobilized HC-HA/PTX3 polarizes LPS and IFN-γ/LPS-activated macrophages toward the M2 phenotype.17,23,31 In short, immobilized HC-HA/PTX3, but not HA, upregulates transcript and protein expression of M2 markers (e.g., IL-10 and TGF-β1) and downregulates M1 markers (e.g., IL-12p40 and TNF-α) in activated RAW264.7 cells (See Figures 2A & B). In addition, such M2 polarization is coupled with notable downregulation of IL-23 (See Figure 2C), which is the cytokine produced by activated macrophages and dendritic cells to activate Th17 cells.32,33

4) HC-HA/PTX3 suppresses activation of CD4+ T cells. Naïve CD4+ T cells can be activated to proliferate and differentiate into Th1, Th2, Th17 or Tregs.34-36 Th1 cells secrete IFN-γ and IL-2 to enhance pro-inflammatory responses.37,38 This action can be downregulated by Tregs that is activated by M2 macrophages.39 Our study shows the soluble HC-HA/PTX3 suppresses the proliferation and production of IFN-γ and IL-2 and expression of T cell activation markers (CD25 and CD69) while significantly promoting the expansion of CD25+/FOXP3+ T cells.31 These data strongly suggest that HC-HA/PTX3 suppresses activation of CD4+ T cells into Th1 cells.

5) HC-HA/PTX3 suppresses the macrophage influx to LPS-elicited corneas and prolongs survival of corneal allografts. While intrastromal injection of LPS elicited notable influx of EGFP+ macrophages to the corneal periphery in Mafia mice from day one to day five,40,41 subconjunctival injection of HC-HA/PTX3 significantly suppresses the infiltrated macrophages. Further, the infiltrated macrophages are polarized towards an M2 phenotype by expressing higher M2 markers (Arg-1 and IL-10) but lower M1 markers (IL-12p35 and IL-12p40).31 Using a murine orthotropic corneal transplantation model,42-45 we have found that subconjunctival injection of HC-HA/PTX3 prolongs the significant survival of allografts compared to PBS injection.31 These data suggest that HC-HA/PTX3 may be used to exert a potent anti-inflammatory action leading to suppression of (alloreactive) immune activation in vivo.

HC-HA/PTX3, a unique matrix component purified from human AM, retains AM’s multifactorial anti-inflammatory actions. It is foreseeable that HC-HA/PTX3 can be used as a new class of biologics to treat ocular inflammatory diseases (e.g., proliferative diabetic retinopathy, trauma and subretinal fibrosis secondary to age-related macular degeneration and other choroidal neovascular processes) as well as similar pathological processes in other parts of the body.  REVIEW


Dr. Tseng is a physician scientist and the chief scientific officer of TissueTech Inc, where both Dr. He and Dr. Zhang serve as research scientists. Contact Dr. Tseng at Ocular Surface Center, 7000 SW 97 Ave., Ste. 213, Miami, Fla. 33173. Phone: (305) 274-1299; fax: (305) 274-1297; e-mail: stseng@ocularsurface.com.

Supported in part by grants from National Institute of Health, National Eye Institute. Additional support is from a research grant from TissueTech Inc. and an unrestricted grant from Ocular Surface Research & Education Foundation, Miami.



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