Tomas Blanco

MMP9 cleavage of the β4 integrin ectodomain leads to recurrent epithelial erosions in mice

Integrin α6β4 is an integral membrane protein within hemidesmosomes and it mediates adhesion of epithelial cells to their underlying basement membrane. During wound healing, disassembly of hemidesmosomes must occur for sheet movement-mediated cell migration. The mechanisms of disassembly and reassembly of hemidesmosomes are not fully understood. The current study was initiated to understand the underlying cause of recurrent corneal erosions in the mouse. Here, we show that in vivo: (1) MMP9 levels are elevated and β4 integrin is partially cleaved in epithelial cell extracts derived from debridement wounded corneas; (2) the β4 ectodomain is missing from sites where erosions develop; and (3) β4 cleavage can be reduced by inhibiting MMP activity. Although β4, α3 and β1 integrins were all cleaved by several MMPs, only MMP9 was elevated in cell extracts derived from corneas with erosions. Coimmunoprecipitation studies showed that β4 integrin associates with MMP9, and protein clustering during immunoprecipitation induced proteolytic cleavage of the β4 integrin extracellular domain, generating a 100 kDa β4 integrin cytoplasmic domain fragment. Confocal imaging with three-dimensional reconstruction showed that MMP9 localizes at erosion sites in vivo where the ectodomain of β4 integrin is reduced or absent. MMP activation experiments using cultured corneal and epidermal keratinocytes showed reduced levels of α6β4 and β1 integrins within 20 minutes of phorbol ester treatment. This report is the first to show that β4 integrin associates with MMP9 and that its ectodomain is a target for cleavage by MMP9 in vivo under pathological conditions.

Involvement of corneal lymphangiogenesis in a mouse model of allergic eye disease.

PURPOSE The contribution of lymphangiogenesis (LA) to allergy has received considerable attention and therapeutic inhibition of this process via targeting VEGF has been considered. Likewise, certain inflammatory settings affecting the ocular mucosa can trigger pathogenic LA in the naturally avascular cornea. Chronic inflammation in allergic eye disease (AED) impacts the conjunctiva and cornea, leading to sight threatening conditions. However, whether corneal LA is involved is completely unknown. We addressed this using a validated mouse model of AED. METHODS Allergic eye disease was induced by ovalbumin (OVA) immunization and chronic OVA exposure. Confocal microscopy of LYVE-1-stained cornea allowed evaluation of corneal LA, and qRT-PCR was used to evaluate expression of VEGF-C, -D, and -R3 in these mice. Administration of VEGF receptor (R) inhibitor was incorporated to inhibit corneal LA in AED. Immune responses were evaluated by in vitro OVA recall responses of T cells, and IgE levels in the serum. RESULTS Confocal microscopy of LYVE-1-stained cornea revealed the distinct presence of corneal LA in AED, and corroborated by increased corneal expression of VEGF-C, -D, and -R3. Importantly, prevention of corneal LA in AED via VEGFR inhibition was associated with decreased T helper two responses and IgE production. Furthermore, VEGFR inhibition led a significant reduction in clinical signs of AED. CONCLUSIONS Collectively, these data reveal that there is a distinct involvement of corneal LA in AED. Furthermore, VEGFR inhibition prevents corneal LA and consequent immune responses in AED.

The cornea has "the nerve" to encourage immune rejection.

‘Supersystem’ is a designation given to a highly integrated life system, which was coined by the late Tomio Tada nearly a decade ago to describe both immune and nervous systems [1]. Even decades earlier, however, were scientists already appreciating the level of interplay that exists between these two supersystems. Work by Paunicka et al now highlight the importance of this relationship in corneal allograft rejection. The cornea is a fascinating model site to explore how the nervous and immune systems interface, as it is arguably the most densely innervated site in mammals. Furthermore, the cornea enjoys immune privilege status, which is a feature that permits spontaneous acceptance in approximately 50% of corneal allografts in certain mouse strain combinations [2]. Paunicka et al now show that the severing of corneal nerves in the graft bed, an unavoidable consequence of penetrating keratoplasty, abolishes immune privilege that would otherwise be enjoyed by subsequent allografts, even in the fellow eye. This perhaps explains why a markedly increased incidence of failure of second grafts is seen in the clinic. The authors came to this conclusion with elegant experimentation. They initially found that placement of an allograft led to the increased tempo and incidence of rejection to a subsequently placed corneal allograft in the same, or fellow eye. Importantly, this pattern was not due to a second-set rejection, as the same result was observed when the subsequent allograft was unrelated to the first. However, this antigen nonspecific increase in rejection was indeed T cell mediated, as subsequent syngeneic grafts survived indefinitely. The root cause of increased rejection turned out to be the nerve injury response, originating from the first transplantation. The same results were recapitulated by simply severing of nerves 360 degrees around the cornea. Remarkably, an allograft placed 60 days after such injury also succumbed to increased immune rejection. Authors conclusively demonstrated that a burst of the neuropeptide substance P, produced as an effect of such nerve severing, caused this heightened state of rejection. Furthermore, they show that substance P disables T regulatory (Treg) suppression, and associated this impairment with increased rejection. How could Tregs be impaired and augment immune rejection to a substance P burst that occurred 60 days prior? The authors are unsure, particularly given a serum half-life of only several minutes. Perhaps one could point to a role for tissue resident macrophages as a possible explanation. Indeed, these mononuclear phagocytes have a long life span of many months to even a year. Furthermore, macrophages express substance P receptors, produce substance P, and are altered by ligation of this neuropeptide [3]. Additionally, macrophages are antigen-presenting cells and can migrate to lymphoid organs, likely in a CCR7 mediated fashion [4]. Importantly, it has been long appreciated that many macrophages reside in close proximity to peripheral nerves [5], consistent in the cornea as well [6]. One can appreciate this in Figure 1, which is an unpublished micrograph generated by our lab demonstrating the proximity of a mononuclear phagocyte with up to 10 distinct nerves in the corneal epithelium. Perhaps one plausible hypothesis to explain the fascinating findings by Paunicka et al is that the unilateral severing of corneal nerves that causes a bilateral substance P burst has a pathogenic imprinting effect on such long-lived macrophages residing proximal to nerves. Thus, upon transplantation, either immediately or long after the substance P burst, perhaps this imprint licenses macrophages to produce additional substance P. If these antigen-presenting cells, laden with alloantigen, could migrate to the lymphoid organ, their substance P may therefore subvert Treg activity and in turn may lead to increased immune rejection. Figure 1 Mononuclear phagocytes are in contact with corneal nerves Irrespective of how substance P produces such a long-lasting ability to augment immune rejection, Paunicka et al forces transplantation immunologists, particularly those studying the cornea, to now consider the interplay of both supersystems as yet another piece of the perplexing puzzle that is immune rejection.