Microglia derived IL-6 suppresses neurosphere generation from adult human retinal cell suspensions

Abstract

Following retinal degeneration or inflammation that disrupts tissue architecture, there is limited evidence of tissue regeneration, despite evidence of cells with progenitor properties in the adult human retina at all ages. With the prospect of tissue/cell transplantation, redressing homeostasis whilst overcoming glial barrier or gliosis remains key to successful graft versus host integration and functional recovery. Activated human retinal microglia (MG) secrete cytokines, including IL-6, which may suppress neurogenesis or cellular (photoreceptor) replacement.

To investigate this hypothesis, adult human retinal explants were cultured in cytokine-conditioned media (TNFα, TGFβ, LPS/IFNγ) to activate microglia in situ. Following culture of retinal explants for 4 days, supernatant conditioned by resulting migrated microglia was collected after a further 3 days and fed to retinal cell suspensions (RCS). Neurosphere (NS) generation and survival analysis was performed after 7 and 14 days in culture, with or without addition of conditioned media and with or without concomitant IL-6 neutralisation. Neurosphere phenotype was analysed by immunohistochemistry and cell morphology.

Migratory MG from retinal explants were activated (iNOS-positive) and expressed CD45, CD11b, and CD11c. LPS/IFNγ-activated MG conditioned media (MG-CM) contained significant levels of IL-6 (1265 ± 143) pg/ml, which inhibited neurosphere generation within RCS in the presence of optimal neurosphere generating N2-FGF2 culture medium. Neutralising IL-6 activity reinstated NS generation and the differentiation capacity was maintained in the spheres that formed. Even in the presence of high levels of IL-6, those few NS that did form demonstrated a capacity to differentiate. The data supports activated MG-derived IL-6 influence retinal cell turnover.

Introduction

The adult human retina and ciliary body contains retinal progenitor cells (RPCs) (Mayer et al., 2003, Mayer et al., 2005, Coles et al., 2004, Yang et al., 2002). Adult human retinal progenitor cells have evidence of a putative capacity to proliferate and differentiate into all retinal cell types, including neurons, glia and photoreceptor cells (Carter et al., 2009, Carter et al., 2007, Barraud et al., 2007, Uchida et al., 2000, Moshiri et al., 2004). Their presence in adult human retina implies the RPCs possess an inherent ability for cellular replacement throughout life (Ziv et al., 2006). This may be explained by the presence of somatic progenitor cells, which unlike stem cells have limited (only when stimulated in the event of tissue damage) asymmetric (certain cells retain parent properties whilst others differentiate into mature cells) proliferative potential but permit cell renewal via differentiation into mature functional cells as we and others have reported (Carter et al., 2009, Coskun et al., 2008, Nickerson et al., 2007, Morshead et al., 1994, Knox Cartright et al., 2007, Sell, 2004). Cellular replacement in the retina may occur in three main contexts: firstly, in the maintenance of normal retina, as part of tissue homeostasis; secondly, in the repair and regeneration that is required after cell loss; and thirdly in the retinal remodeling that occurs in response to retinal injury, degeneration or inflammation, when tissue structure is disrupted or lost. The latter involves the loss of polarity signals and extracellular matrix enabling cells to integrate precisely where they are needed. Consequently these conditions are more often associated with scar formation (Mayer et al., 2003).

Myeloid-derived, peri-, para- vascular and parenchymal retinal microglia (MG) are constantly replaced from bone marrow (Xu et al., 2007). They are activated during degeneration and inflammation and appear to have a pivotal role in maintaining the steady state (Dick, 2008, Carter and Dick, 2004, Xiao and Link, 1999, Chen et al., 2002, Dick et al., 2003, Broderick et al., 2002, Ponomarev et al., 2005). The pleiotropic functions of retinal microglia are reflected by their cell surface phenotype, which includes differential extent of expression of CD45 (leucocyte common antigen), CD11b, CD11c, CX3CR1, MHC class II (higher expression in paravascular MG) reflecting degrees of activation, as well as components of phagocytic machinery such as CD68 (macrosialin, lysosomal membrane marker), Mac S22 (a macrophage antigen found on only 10% of human retinal MG) and sialoadhesin (macrophage-restricted cell surface sialic acid receptor protein) (Provis et al., 1995, Albini et al., 2005, Langmann, 2007, Albright and Gonzalez-Scarano, 2004, Dick et al., 1995, Becher and Antel, 1996, Gregerson et al., 2004, Gregerson and Yang, 2003, Wong et al., 2005, Chen et al., 2002, Lemstra et al., 2007, Hughes et al., 2003). In normal retina these cells are quiescent and anti-inflammatory (Ponomarev et al., 2005). However, previous studies have shown retinal MG to have the capacity to be activated, migrate and phagocytose. In addition, retinal MG, like activated macrophages/monocytes (Mertsch et al., 2001, Carter and Dick, 2003, Carter and Dick, 2004, Egensperger et al., 1996) generate a plethora of cytokines and growth factors including, FGF2, chemokines (CXCR4), cytokines (IL-1β, IL-6, IL-8, IL-10, IL-12, IL-17), and up-regulate cytokine receptors (IL-6R, IL-8R, IL-10R, IL-12R), as well as TNF-α, prostaglandins, nitric oxide (NO) and neurotoxins (neurotoxic amine and quinolinic acid) (Lee et al., 2002, Nagai et al., 2001, Yoshida et al., 2004, Ehrlich et al., 1998, Giulian et al., 1996, Giulian et al., 1990). Retinal microglia are able to drive inflammation and tissue destruction as well as regulate repair (Broderick et al., 2002, Dick et al., 2003), which suggests a pivotal role in co-ordinating tissue responses and consequently makes them a key target for disease-modifying treatment in the future. Human retinal microglia are activated in response to pro-inflammatory signals (LPS/IFNγ, TNF-α) and anti-inflammatory (TGF-β) stimuli (Carter and Dick, 2003, Langmann, 2007) that regulate their proliferation, migration and activation status. Retinal microglia influence neuronal survival and propagation (replacement of neurons by retinal progenitors and cell division of migrated cells through tissue) as well as active phagocytosis of dying photoreceptor cells during development or in the adult when inflammation or degeneration ensues (Xiao and Link, 1999, Egensperger et al., 1996).

There is evidence that microglial cells influence CNS stem/progenitor cell populations (Ziv et al., 2006). In rodents, IL-6 and LIF (part of a family of signalling molecules) act via gp130/STAT3 and influence CNS neural PC turnover and differentiation (Nakanishi et al., 2007). Furthermore, IL-6, CNTF and LIF modulate retinal cell survival and differentiation (Nakanishi et al., 2007, Nishimoto, 2006). LIF regulates neural and RPC ability to proliferate and differentiate, and form neurospheres from both primary and CD133-enriched cells (Corti et al., 2007, Barraud et al., 2007, Uchida et al., 2000, Carter et al., 2009).

Utilising a simulated activated retinal environment, interaction of putative retinal neural progenitor cells (the presumed source of retinal cell replacement) and activated MG (secreting IL-6) was assessed. The role of IL-6 in intercellular crosstalk between retinal cells and MG cells is explored within retinal cell suspensions (RCS) by assessing their capacity to generate neurospheres (NSs).