An Immunomodulating Peptide With Potential to Counteract Endometriosis

Endometriosis is an oestrogen‐dependent, chronic inflammatory disease that affects approximately 10% of women and why it develops in only a fraction of women following retrograde menstruation is not known. Despite decades of animal‐based and human studies, it also remains unclear how the endometriotic cellular and cytokine milieu supports growth of ectopic endometrial tissue. Aberrant immune responses in the peritoneal cavity considered responsible for diminished lysis of ectopic endometrial tissue are thought to include the presence of immature dendritic cells (DCs) [ 1 ], dysfunctional natural killer (NK) cells [ 2 , 3 , 4 ] including less cytotoxic CD8+ T cells [ 5 ] and cytokine imbalance involving the interleukin‐12 (IL‐12) isoforms [ 2 ]. The heterodimeric IL‐12 isoform, IL‐12p70, is comprised of p40 and p35 subunits whereas IL‐12p40 exists either as a p40 monomer or p40 homodimer. In the seminal work conducted by Mazzeo and colleagues 27 years ago [ 2 ], levels of p40 in peritoneal fluid of endometriosis patients were found to correlate with more severe disease. These investigators showed that IL‐12p70‐mediated lysis of human endometrial cells by NK cells was inhibited in the presence of the IL‐12p40 monomer (p40), thus highlighting the importance of the IL‐12p70: IL‐12p40 ratio in the cytotoxic response of NK cells towards endometrial targets [ 2 ]. Notably, in contrast to IL‐12p40 which binds only to the high affinity IL‐12Rβ1 chain of the IL‐12 heterodimeric receptor complex, IL‐12p70 must also bind to a β2 subunit (IL‐12Rβ2) for signalling via IL‐12p70 to occur [ 6 ]. However, IL‐12p40 is normally expressed in large excess in immune cells compared to IL‐12p70 [ 7 ] as is expression of the β1 receptor chain in NK cells compared to the β2 subunit in the absence of T cell receptor activation [ 8 ]. Taken together, skewed IL‐12 signalling in NK cells towards the IL‐12p40 axis could be considered a functional deficit that contributes to diminished lysis of endometriotic lesions. This is reflected in diminished cytotoxicity mediated by NK cells isolated from either blood or peritoneal fluid of endometriosis patients compared with healthy individuals when co‐cultured with the erythroleukemic cell line, K562, that is devoid of classical HLA Class I and Class II molecules [ 4 ]. Hence, K562 cells are less effectively lysed by cells obtained from endometriosis patients compared to healthy individuals [ 4 ]. Cytokines other than the IL‐12 isoforms also play key roles in regulating progression of endometriotic lesions. For example, IL‐12p40 is produced mainly by DCs and acts as a potent inducer of Type II interferon, IFN‐γ, in T cells that involves a positive feedback loop [ 7 ]. Elevated levels of IFN‐γ in endometriotic tissues compared with normal endometrium appear to drive endometriosis progression [ 9 , 10 ]. In endometriosis patients, levels of IFN‐γ are significantly elevated in the peritoneal fluid of endometriosis patients compared with healthy women and IFN‐γ fails to induce apoptosis in endometrial stroma cells [ 10 ]. The expression of IFN‐γ is also highly increased within implantation sites in murine models of endometriosis and it has been suggested that IFN‐γ enhances the invasive activity of ectopic endometrial lesions [ 10 ]. In murine models, IL‐12p70‐mediated cytotoxicity by NK cells against endometrial tissues has been shown to depend on the presence of interleukin‐2 (IL‐2). For example, blocking IL‐2‐mediated signalling with a neutralising antibody against the IL‐2 receptor subunit, IL‐2Rβ, prevents 12p70‐mediated suppression of endometriosis [ 11 ]. Moreover, IL‐2 tested in rat models of endometriosis has yielded promising results [ 12 ]. IL‐2 drives production of lytic molecules [ 8 ] and reduced levels of granzyme B and perforin secreted by NK cells contribute to diminished cytotoxicity against refluxed endometrial fragments in the peritoneal cavity [ 3 ]. Furthermore, administration of the Type I IFN (IFN‐β) has been shown to reduce the size of endometriosis implants and IFN‐β dramatically inhibits endometrial stromal cell migration via suppression of the extracellular signal‐regulated kinase (ERK) pathway [ 10 ]. In contrast to immature DCs, DC maturation secondary to interactions between CD40 ligand‐expressing T cells and CD40 expressed on DCs results in IL‐12p70 production [ 7 ]. Given the inhibitory effect of p40 on IL‐12p70‐mediated lysis of endometrial lesions [ 2 ] and the presence of immature IL‐12p40‐producing DCs in peritoneal fluid of endometriosis patients, it is not surprising that immature DCs have been implicated in endometriosis [ 1 ]. Furthermore, the feedback loop between IL‐12p40 and IFN‐γ produced by DCs and T cells, respectively [ 7 ], sustains IL‐12p40 production which is dependent upon ERK signalling [ 13 ]. Hence, the mitogen‐activated protein kinase (MAPK) signalling pathway has been identified as an important contributor to the microenvironment underlining progression of endometriosis and levels of phosphorylated ERK1/2 are significantly higher in endometriotic stromal cells than endometrial stromal cells [ 14 ]. Importantly, in contrast to IL‐12p40, induction of the p35 subunit is ERK‐independent [ 15 ]. We have previously reported that a 15 mer peptide derived from the β6 integrin subunit, that is, RSKAK WQTGT NPLYR, binds to ERK [ 16 ] and lack of this sequence in the β6 integrin cytoplasmic domain markedly suppresses cancer growth in vivo [ 16 ]. Moreover, exposure of cancer cells to a non‐naturally occurring deletion variant of this peptide, that is, RSKAKNPLYR that lacks the amino acids WQTGT (underlined above), also binds to ERK and inhibits cancer cell proliferation in vitro as effectively as the 15 mer sequence at relatively high micromolar concentrations [ 17 ]. Conjugation of the smaller peptide to hyperbranched lauric acid residues to minimise intracellular enzymatic degradation of the peptide has led to the discovery of a compound, designated IK14004, that induces immunomodulating effects in human immune cells in vitro at nanomolar concentrations and suppresses tumour growth in murine models [ 7 , 8 ]. Although antigen‐presenting cells and peripheral blood CD4+ T cells are known to express IL‐12, IK14004 is the only agent described to date that induces IL‐12p70 protein expression in human T cells [ 7 ]. The lipidic peptide destabilises immature monocyte‐derived human DCs in vitro leading to suppression of IL‐12p40 production by DCs and inhibition of IFN‐γ expression in T cells [ 7 ]. In contrast, production of IL‐12p70, but not IL‐12p40, by CD3+ T cells is enhanced [ 7 ]. Notably, T cell secretion of IL‐2 and IFN‐β also increases upon exposure to peptide at low‐to‐mid nanomolar concentrations [ 7 , 8 ]. Besides reduced lysis of endometriotic lesions by dysfunctional NK cells, the proportion of CD8+ T cells is reduced in endometriotic lesions associated with reduced cytotoxicity of ectopic CD8+ T cells [ 5 ]. CD8+ T cells and NK cells are activated in the presence of IL‐12p70 and expression of the β2 chain of the IL‐12p70 receptor complex is the limiting factor that determines responsiveness to IL‐12p70 [ 7 ]. Exposure of NK and CD8+ T cells to IK14004 induces a proportionately greater increase in expression of the β2 receptor subunit above basal levels compared with the β1 chain, which is linked to enhanced IFN‐β production [ 8 ]. Taken together with enhanced availability of IL‐12p70, this indicates IK14004‐mediated skewing of IL‐12 signalling in cytotoxic lymphocytes towards the IL‐12p70 axis. Co‐cultures of peripheral blood mononuclear cells (PBMCs) with the K562 cell line are a surrogate tool that provides an indication of the immunocompetency of cytotoxic lymphocytes to lyse endometrial lesions [ 4 ] and IK14004 enhances K562 lysis in the presence of PBMCs isolated from healthy donors [ 8 ]. Besides suppression of NK cell‐mediated lysis of ectopic endometrial tissue, decreased CD8+ T cell‐mediated cytotoxicity also plays a role in endometriosis [ 5 ] and whether IK14004 activates both CD8+ T cells and NK cells in the K562 assay is not known. The capacity of CD8+ T cells to kill cancer cells, for example, K562 cells that lack MHC‐I expression, is dependent upon interactions between the natural killer group 2D (NKG2D) receptor expressed on CD8+ T cells and NKG2D ligands [ 18 ] and IK14004 induces NKG2D expression in CD8+ T cells associated with increased granzyme B expression [ 7 ]. Interestingly, in peritoneal fluid from endometriosis patients, expression of NKG2D in NK cells is decreased [ 19 ]. NK cell‐mediated killing of K562 cells is also NKG2D‐dependent and IK14004 induces NKG2D expression in NK cells [ 8 ]. Importantly, IL‐2 enhances expression of NKG2D in NK cells [ 8 ] and IL‐2‐activated NK cells elicit potent cytotoxicity against co‐cultured ectopic endometrial stromal cells [ 20 ]. Given that IK14004 induces IL‐2 production by CD4+ T cells [ 8 ], this raises the possibility that peptide plus recombinant IL‐2 could enhance NK cell‐mediated cytotoxicity further in either an additive or synergistic manner. In summary, the pelvic cytokine milieu and NK/CD8+ T cell receptor activity appear to dictate immunocompetency required to clear ectopic endometrial lesions from the peritoneal cavity. Given that the pathogenesis of pelvic endometriosis remains unclear, the powerful biological implications of IK14004 are that fine‐tuning of key cytokine components underlying endometriosis may now be possible. For example, production of IL‐12p40 from IL‐12p70 can be uncoupled as can production of Type I from Type II IFNs. This is relevant when seeking to determine whether chronic pelvic pain in women is caused by underlying endometriosis. For example, failure of IK14004 to induce these uncoupled cytokine effects and/or enhance expression of NKG2D in cell preparations from peripheral blood may have diagnostic relevance in women experiencing chronic pelvic pain. Taken together, this could facilitate early diagnosis of endometriosis in a non‐invasive manner and may even have therapeutic benefit in combination with standard medical treatment.

The author declares a conflicts of interest as a board member of InterK Peptide Therapeutics Limited.