Lack of inflammatory gene expression in bats: a unique role for a transcription repressor

Abstract

In recent years viruses similar to those that appear to cause no overt disease in bats have spilled-over to humans and other species causing serious disease. Since pathology in such diseases is often attributed to an over-active inflammatory response, we tested the hypothesis that bat cells respond to stimulation of their receptors for viral ligands with a strong antiviral response, but unlike in human cells, the inflammatory response is not overtly activated. We compared the response of human and bat cells to poly(I:C), a viral double-stranded RNA surrogate. We measured transcripts for several inflammatory, interferon and interferon stimulated genes using quantitative real-time PCR and observed that human and bat cells both, when stimulated with poly(I:C), contained higher levels of transcripts for interferon beta than unstimulated cells. In contrast, only human cells expressed robust amount of RNA for TNFα, a cell signaling protein involved in systemic inflammation. We examined the bat TNFα promoter and found a potential repressor (c-Rel) binding motif. We demonstrated that c-Rel binds to the putative c-Rel motif in the promoter and knocking down c-Rel transcripts significantly increased basal levels of TNFα transcripts. Our results suggest bats may have a unique mechanism to suppress inflammatory pathology.

Caution: Choice of Fixative can Influence the Visualization of the Location of a Transcription Factor in Mammalian Cells

“…Although IRF3 has been studied extensively in rodents and primates, not much is known about bat IRF3. We have previously detected big brown bat ( Eptesicus fuscus ) IRF3 transcripts and studied the upregulation of IRF3 transcripts in response to mimic viral challenge in both human and bat cells (see supplementary information in [8]). While optimizing the detection of bat IRF3 protein by immunofluorescence, we discovered that the location of this transcription factor varied depending on whether we used 100 methanol or 10 NBF as fixative.…”

“…These include viruses that cause diseases such as severe acute respiratory syndrome, Middle-East respiratory syndrome (MERS), Marburg hemorrhagic disease, and Hendra and Nipah respiratory and neurological syndromes. These viruses, or bat viruses closely related to them, do not appear to cause noticeable morbidity or pathology in their natural bat hosts (reviewed in [6,7]), possibly due to unique mechanisms to dampen potentially harmful inflammation in response to viral infections [8] and a constitutively active antiviral interferon response [9]. …”

“…Media used for these cell types have been previously described [8]. The chambers were incubated at 37C in a humidified incubator overnight.…”

“…All samples were treated and analyzed by immunoblotting as previously mentioned [8]. Rabbit anti-human-IRF3 (Abcam; Catalogue number: ab68481; RRID: AB_11155653) and mouse anti-GAPDH (EMD Millipore, USA; Catalogue number: AB2302; RRID: AB_10615768) primary antibodies were used at a dilution of 1/1000.…”

“…Slides that were simultaneously fixed in methanol followed by 10 NBF were also permeabilized using 0.2 TritonX-100 for consistency. All cells were washed twice and then stained for IRF3, Lamin B1, GAPDH and nuclear DNA using a protocol previously described [8]. 150l of solution was used for staining and subsequent washes in each chamber.…”