Inflammatory gene expression in response to sub-lethal ricin exposure in Balb/c mice
Introduction
Ricin is a major protein produced in the seeds of the castor oil plant, Ricinus communis, and forms a dimeric 65 kDa toxin comprising an A and B chain joined by a disulphide bond. The toxin binds to cell surface carbohydrates by attachment to lectin moieties in the B chain subunit and is transported into the cell via endocytosis and retrograde translocation via the Golgi apparatus (Roberts and Smith, 2004, Liu et al., 2006). The holo-toxin is a type II ribosome-inactivating protein (RIP) (Barbieri et al., 1993), which cleaves adenine 4324 of the 60S subunit of ribosomal RNA leading to arrested protein synthesis and ultimately cell death (Jiminez and Vasquez, 1985).
Ricin has been designated a Schedule 1 chemical under the terms of the 1993 Chemical Weapons Convention (OPCW, 1993). Its toxicity is dependant on both dose and route of exposure and severe toxic effects are manifested following ingestion, intramuscular injection or inhalation. Inhalational exposure of ricin has been reported to cause severe lung damage as early as 15 min post-inhalation (Assaad et al., 1991), with definite pathological changes observed by electron microscopy from around 12 h after exposure (Brown and White, 1997). The lungs are the most likely route of exposure in both terrorist and military situations involving chemical agents, leading to fatal or severely incapacitating acute lung injury. Previous work on a rat model of ricin inhalation exposure (Griffiths et al., 1995) has reported the probit curve of ricin (var. zanzibariensis), which indicates a very steep slope when comparing dose versus mortality. These small changes in dose leading to large changes in the response to ricin challenge further complicate the overall picture of mortality following exposure. In military or civilian scenarios involving ricin dissemination by aerosol generation, the most likely outcome of an attack would be a large scale poisoning at a low dose. The individuals exposed to a low dose will inevitably pose a substantial logistical burden on medical care systems. These individuals are also those most likely to be aided by administration of a medical countermeasure.
The pathology of lung damage in association with ricin poisoning has been extensively reviewed elsewhere (Brown and White, 1997, Griffiths et al., 2007, Wannemacher and Anderson, 2006). In brief, damage is confined to the lung and initiated with perivascular oedema progressing to include intra-alveolar regions at later time points. This is followed by the influx of inflammatory cells such as macrophages and polymorphonuclear leucocytes and by apoptotic processes at the alveolar surface. These pathological changes may be exacerbated by the inflammatory response mounted by the host resulting in further damage to the lung. The combined effects lead to flooding of the lung and therefore reduced pulmonary function which, in the most severe of cases, can lead to death. In the event of survival (especially relevant to lower dose ricin exposures) lung damage is resolved and the main repair activities appear to be hyperplasia of type 2 epithelial cells along with clearing of the oedema fluid. These repair mechanisms lead to restoration of the lung and subsequent relief of clinical signs, although pathologically the lung never returns to a completely normal state, due to fibrosis.
As well as direct lung damage, such as perivascular, interstitial and alveolar pulmonary oedema (Griffiths et al., 1999), exposure to ricin also induces an acute inflammatory response mediated primarily through the release of cytokines and chemokines, through a variety of cellular pathways (Gonzalez et al., 2006, Yamasaki et al., 2004). This inflammatory response alerts the innate immune system to the lung damage itself but also can act through the same mediators (e.g. TNFα and IL1β) to exacerbate the injury (Licastro et al., 1993). An extensive array of mediators has been associated with the influx of neutrophils in response to an inhaled toxin including RANTES, MIPs, MCPs and ILs 1, 6, 10 and 12 (Larsson et al., 2000). Whilst some mediators are involved in initiation, maintenance and exacerbation of the inflammatory responses, others are involved in its attenuation and resolution.
Many conditions of the lung, such as COPD and asthma, are also associated with abnormal inflammatory responses. Although these may differ in the inflammatory cells and mediators which become involved, it is now becoming clear that, in some instances, the boundaries between disease responses are not distinct. This leads to new implications for therapeutic intervention where commonalities in response can be exploited (Barnes, 2008). There are numerous examples in the literature focusing on disease process molecular signals, and their potential as targets for therapeutics, towards disease states such as COPD (Mroz et al., 2007) and ARDS (Greenberger, 2008). Common processes across inflammatory lung damaging insults could, theoretically, be targeted towards a generic therapeutic to improve the outcome arising from the injury. A clearer understanding of the processes involved in ricin poisoning would assist in targeting therapeutic strategies to such insults. Data generated could also be exploited in strategies other than medical countermeasures, such as diagnostics and disease kinetics of inflammatory lung damage.
The present study investigated the effects of inhalation exposure to a sub-lethal dose of ricin, in contrast to the majority of previous studies, which have focused on identifying and counteracting lethal and supra-lethal exposure levels. The present study utilised a low dose murine exposure model using the Balb/c mouse. This species was chosen to establish the model because of the wealth of information currently available on the immunological profile of this murine strain and the fact that the genome has been fully sequenced (Waterston et al., 2002) as well as to facilitate direct comparison with a previous microarray study using a higher dose (1 LD50) (DaSilva et al., 2003). Studies in a rat model exposed to LCt30 of ricin showed signs of lung regeneration via pulmonary epithelial hyperplasia at 96 h post-exposure (Griffiths et al., 1995). The present study analysed a 96-h time course following a sub-lethal challenge with ricin, due to this progress towards resolution of injury. This sub-lethal dose was considered important in order to study healing responses throughout a reasonable recovery period.
The complex nature of the host response to ricin means that it is essential to know the mediators involved and their temporal pattern of release, to begin to search for therapeutic drugs and administration strategies. To gain an insight into the expression of pulmonary genes in response to a sub-lethal ricin exposure a customised murine cDNA microarray approach was undertaken. A custom designed microarray platform consisting of 1509 selected genes associated specifically with immune, inflammatory and healing responses was selected to probe the genetic response to ricin in the whole lung of the Balb/c mouse model. A temporal expression pattern across a 96-h time course was studied. Normalisation against sham-exposed tissue enabled the response due solely to agent to be studied and to be correlated with histopathological analysis.
The work presented here describing the host response to ricin is part of a wider study which seeks to identify common genetic elements in the host response in the Balb/c mouse lung to a range of lung damaging agents. The elucidation of genes changing in response to ricin will enable a greater understanding of the mechanisms of response to ricin challenge and offers the potential to augment the development of therapeutics to ricin.
Section snippets
Animals
Balb/c female mice (Charles River Laboratories Ltd., Margate, Kent, UK) weighing 19.6 ± 0.9 g prior to exposure were habituated to the experimental animal unit for 1 week prior to use in the study. Environmental conditions were maintained at 21 ± 2 °C and 55 ± 10% humidity. Lighting was set to mimic a 12/12 h dawn to dusk cycle. Mice were housed in polycarbonate cages (six animals per cage) with steel cage tops with corncob bedding (International Product Supplies, Wellingborough, UK). The mice were fed
Microarray
Following statistical analysis of the data by ANOVA, a false discovery rate correction was applied (Benjamini and Hochberg) to adjust for multiple testing (Benjamini and Hochberg, 1995). Out of the 1509 probes tested, a total of 685 genes were statistically significant from controls in response to ricin, across the 96-h time course (p ≤ 0.05). This indicates the number of genes significantly altered by over 2-fold following normalisation to the control (saline exposed) samples. The top 20 genes
Discussion
Histopathological analysis following low dose exposure to ricin is consistent with that observed in previous studies (Assaad et al., 1991, Brown and White, 1997, Griffiths et al., 1995). Essentially, the development of lesions was noted from the 7-h time point but most damage was established at 24 h after ricin challenge with the onset of alveolar oedema, accompanied by the infiltration of inflammatory cells and haemorrhage progressing through to 48 h. Healing responses were apparent in the
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors would like to thank Mr. David Cook and Mrs. Rachael Whiting for their assistance with the experimental methods associated with RNA extraction and microarray, also Mrs. Janet Platt and Mr. Chris Taylor for their help in preparation of the histopathology samples and images. The Veterinary Pathology Laboratories supplied expert histopathological analysis with special thanks to Javier Salguero DVM, PhD, MRCVS. The inhalational exposures were carried out by Dr. Gary J. Phillips and
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