Our studies demonstrate an unexpected role for commensal bacteria in calibrating the responsiveness of antiviral immunity. ABX-mediated alterations of commensal bacteria compromised innate and adaptive immune responses after systemic or respiratory viral infection. Severe defects in the adaptive immune response to LCMV and influenza virus as well as poor control of viral replication pointed toward early defects in the initiation of antiviral responses. Indeed, genome-wide transcriptional profiling and functional assays uncovered a global defect in antiviral responsiveness of macrophages isolated from ABX mice. Upon deliberate manipulation of commensal bacteria, expression of antiviral defense genes and interferon responsive pathways were altered in the steady state. For rapidly replicating viruses, such a delay in initiating antiviral pathways and activating downstream events such as humoral and cell-mediated adaptive immune responses can have dramatic consequences leading to failure to control infection, increased host morbidity, and mortality. Together, these data suggest a model in which signals from commensal bacteria calibrate the activation threshold of innate antiviral immune responses.
Commensal bacterial communities modulate immune cell homeostasis and disease by providing either immunoregulatory or proinflammatory signals. For example, polysaccharide A, isolated from Bacteroides fragilis
, can reduce the severity of intestinal inflammation in two models of IBD (Mazmanian et al., 2008
). Conversely, commensal bacteria can also boost immune responses against mucosal infections (Benson et al., 2009
; Hall et al., 2008
; Ichinohe et al., 2011
; Ivanov et al., 2009
). These studies provoke the hypothesis that commensal-derived signals might influence the systemic immune response to infection. The present study demonstrates that commensal bacteria influence the activation threshold of broadly used innate antiviral response pathways such as the IFN signaling pathway. Induction of a type I IFN response is fundamental and critical for defense against the majority of viruses (Sen, 2001
). Macrophages isolated from ABX mice, however, displayed major defects in expression of key interferon-stimulated response genes even prior to viral exposure compared to macrophages from CNV mice. Reduction in steady-state transcription of antiviral pathways was associated with impaired responsiveness to type I and type II IFNs or virus.
Iwasaki and colleagues observed that commensal bacteria can influence inflammasome activity, an innate signaling pathway involved in responses to bacteria, cytosolic oligomers, and a subset of viruses (Ichinohe et al., 2011
; Lamkanfi and Dixit, 2011
). In addition, two recent reports identified a fundamental interaction between intestinal commensal bacteria and enteric viruses in which virus can utilize bacterial products to enhance infectivity (Kane et al., 2011
; Kuss et al., 2011
). These reports highlight the dynamic interrelationship between viral pathogens, commensal bacteria, and the immune system. Our results reveal a previously unrecognized interplay between commensal bacteria and antiviral interferon signaling pathways in which low-level tonic signaling by commensal bacteria regulates the steady-state readiness of hard-wired antiviral pathways in macrophages.
Tonic signaling has been proposed as a mechanism to maintain optimal responsiveness of signaling pathways in other immunologic settings (Macia et al., 2009
). For example, naive T cells use tonic signals from low-affinity interactions with self-MHC to regulate homeostasis and optimal dynamic responsiveness upon engagement of cognate antigen (Takeda et al., 1996
; Tanchot et al., 1997
). In this current study, tonic signaling was dependent on commensal-derived signals to maintain the fitness of antiviral pathways in macrophages. Although the potential impact of antibiotic treatment on the host virome is largely unexplored, the most direct interpretation of our data is that commensal bacteria calibrate the threshold of innate immune activation to viral infections and suggest steady-state innate immune crosstalk. Such crosstalk can occur in other settings. For example, latent viral infections can render mice less susceptible to bacterial challenge, an effect attributed to basal macrophage activation (Barton et al., 2007
). Conversely, the bacterial species, Wolbachia
, confers protection against viral infections in Drosophila
(Teixeira et al., 2008
). In addition to antibacterial defense genes, bacterial-derived LPS-TLR4 signaling can upregulate transcription of antiviral genes (Amit et al., 2009
; Doyle et al., 2002
). In the case of LPS-TLR4 signaling, antiviral gene expression is initially induced, but rapidly limited by the polycomb repressor Cbx4 (Amit et al., 2009
). This latter observation suggests a potential explanation for the commensal-antiviral immune fitness axis at the transcriptional level. Induction of transcription followed by repression might maintain key antiviral genes in a state of poised transcriptional regulation, rather than a repressed or inactive state. Transcriptional poising, or the presence of both activating and repressive chromatin, enables more efficient transcriptional induction upon exposure to a true inducer of the gene of interest (Cuddapah et al., 2010
). This state of transcriptional equilibrium provided by tonic commensal stimulation may enable rapid induction of antiviral defense genes upon infection. Examples of this type of regulation exist in other biological systems such as the yeast Hog1-MAPK pathway (Macia et al., 2009
). Our results suggest that commensal bacteria provide such a signal to maintain antiviral innate immune pathways in a state of optimal readiness, allowing dynamic and robust responses upon challenge by viral infections.
It was remarkable that macrophages isolated from ABX mice prior to viral infection also displayed less detectable in vivo pSTAT1 compared to macrophages from naive CNV controls. Commensal-derived signals may induce tonic, low-level STAT1 activation in the steady state, which could be a key contributing factor to basal induction of antiviral defense genes prior to infection. The mechanisms through which commensal-derived signals stimulate immune cells in the periphery are poorly understood. One possibility is that peripheral immune cells are directly exposed to bacterial microbes or their products. Small numbers of live commensal bacteria can be found in the Peyer’s patches and mesenteric lymph nodes of mice in the steady state, and there is evidence that absorbed commensal products circulate throughout the host (Clarke et al., 2010
; Macpherson and Uhr, 2004
). Thus, direct interaction between peripheral immune cells and bacterial products is plausible. Alternatively, commensal bacteria may act indirectly on peripheral immune cells via responses evoked from epithelial or other mucosal-associated stromal cells (Artis, 2008
). Defining the potential pathways involved in microbial sensing by the peripheral immune system will be crucial for understanding how microbial crosstalk influences immune cell homeostasis and host protective immunity.
Modulating commensal bacterial communities has therapeutic potential. For example, probiotic treatments can ameliorate intestinal inflammatory diseases (Sartor, 2004
) and success of bacteriotherapy in cases of viral gastroenteritis demonstrates the potential use of probiotics as a treatment strategy to combat viral infection (Fang et al., 2009
; Szajewska and Mrukowicz, 2005
). Further, prophylactic probiotic administration can limit the duration and severity of respiratory viral infections in human subjects, suggesting that the beneficial effects of probiotics on antiviral immunity are not limited to the gastrointestinal tract (de Vrese et al., 2006
). Despite many recent advances in defining the diverse and dynamic microbiome in humans and animal models of human disease, it is unclear which bacterial species or microbial products are associated with the beneficial antiviral effects of commensal bacteria observed in this study. It will be important to define the commensal bacterial species and signals that elicit these host protective effects. Such studies could lead to new approaches for therapeutically administering commensal bacteria or commensal-derived products and selectively manipulating host protective immunity.