pathogens have developed a number of strategies to evade recognition and clearance by the host immune system .
one such strategy , which exists across the microbial taxonomic spectrum , is to invade and establish a unique intracellular niche permissive for microbial replication within host cells . to survive within the host intracellular environment ,
many pathogens employ virulence factors that manipulate host cell processes , such as protein translation and membrane trafficking , to allow for the acquisition of nutrients necessary for growth and replication .
conversely , the innate immune system ( and eventual effector cells of the adaptive immune system ) is capable of recognizing the presence of an intracellular microbial infection and mounting a variety of responses aimed at defeating the pathogen .
examples of recognition strategies include the ligation of host cytosolic pattern recognition receptors ( prrs ) by evolutionarily conserved pathogen - associated molecular patterns ( pamps ) introduced into the host cytosol by intracellular pathogens , or the detection of  patterns of pathogenesis ' , such as the recognition of viral - mediated downregulation of major histocompatibility class i protein expression on the cell surface by natural killer cells .
however , many pathogens have evolved to evade immune detection , as exemplified by the alteration or downregulation of bacterial flagellin expression to evade recognition by the innate immune receptors toll - like receptor ( tlr ) 5 and nlr family , apoptosis inhibitory protein 5 ( naip5 ) . at the expense of additional energy and genomic utilization ,
it has long been known that one of the most potent downstream outcomes of innate immune recognition of intracellular infection involves the rapid production of pro - inflammatory cytokines , such as type i interferons ( ifns ) , interleukin 12 ( il-12 ) and tumor necrosis factor ( tnf ) , which are important for antimicrobial defense .
the prevailing dogma is that these cytokines are produced by directly infected cells in response to prr ligation to alert neighboring , uninfected cells to the presence of a pathogen .
however , it has also long been known that many intracellular pathogens deploy a number of strategies , such as blocking mitogen - activated protein kinase ( mapk ) and nuclear factor-b ( nf-b ) signaling or host translation , to silence this wave of signals in the infected cell and prevent the subsequent propagation of cytokine - mediated immune responses .
thus , it remained unclear how , in the face of immune silencing , the host was capable of initiating and amplifying these signaling and cytokine cascades , as the recognition and response events were thought to take place in cis within an infected cell .
however , with more recent technical advances , particularly those that allow for the study of host : pathogen interactions with single - cell resolution , scientists have begun to appreciate how and where these early cytokines are made .
observations in these studies have led to novel insight into the role of uninfected bystander cells in the primary immune activation events immediately following infection .
we would like to here define bystander cells in the context of innate immunity as uninfected , neighboring cells ( although not necessarily adjacent to or in contact with the infected cell in three - dimensional organotypic space ) , which signal to the immune system , even in lieu of direct pathogen recognition , in a process known as bystander activation . in this model , bystander cells , which may or may not be of the same cell type as the infected cell , produce cytokines upon receiving indirect pathogen recognition signals or microbial - derived products from the infected cell , thus enabling bystander cells to bypass pathogen - mediated attenuation of innate immune signaling within the directly infected cell .
intercellular communication between infected and bystander cells can involve either direct cell  cell contact or soluble signals that act at a distance .
the following sections provide examples of bystander activation in infection models of viral , bacterial and protozoan pathogens , and hosts ranging from drosophila to humans .
these diverse examples serve to support the concept of bystander activation as a critical evolutionary adaptation in metazoan innate immunity .
many of these prrs sense foreign nucleic acids and trigger the production of type i ifns . however , many viruses have evolved virulence factors that antagonize type i ifn production by infected cells .
thus , it is unclear how an effective type i ifn response can be generated during viral infection .
a study utilized an ifn - sensitive response element  green fluorescent protein ( gfp ) reporter cell line that specifically reports activation of the transcription factor ifn regulatory factor ( irf ) 3 rather than type i ifn signaling to probe irf3-dependent responses at the single - cell level . using fluorescence microscopy , this system revealed that the transfection of fluorescently labeled dna complexes into cells induced irf3-dependent reporter expression in both transfected and neighboring untransfected cells .
furthermore , clusters of transfected and untransfected bystander cells produced the majority of antiviral cytokines , such as tnf and ifn , following nucleic acid stimulation .
induction of antiviral responses in bystander cells required cellular contact via gap junctions , which are intercellular channels composed of oligomerized connexin proteins .
the precise molecules communicated through gap junctions and responsible for bystander activation were not identified , in part , because the molecular mechanisms underlying immune sensing of cytosolic dna were poorly understood at this time .
amp synthase ( cgas ) is a key immune sensor critical for host detection of cytosolic dna , both self and foreign . upon binding dna
adenosine monophosphate ( cgamp ) , which binds to the endoplasmic reticulum - resident adapter protein sting ( stimulator of ifn genes ) , thus leading to irf3 activation and subsequent induction of type i ifns .
recently , cgamp was shown to behave as a secondary messenger and be transmitted via gap junctions to activate bystander cells in an in vitro model of vaccinia virus infection ( figure 1 ) .
fluorescence microscopy of cells infected with a gfp - tagged vaccinia strain revealed that the activation of sting by cgamp took place not only in virally infected cells , but also in neighboring bystander cells .
therefore , the ligation of sting in uninfected cells by cgamp produced in infected cells represents a means of bystander activation that circumnavigates the canonical , cell - intrinsic signaling cascade that is thought to occur upon prr engagement .
activation of these bystander cells led to enhanced resistance to viral infection due to induction of antiviral genes .
thus , cell  cell transmission of cgamp via gap junctions enables host cells to mount a successful antiviral response despite viral evasion strategies .
in addition to transmission of cgamp via gap junctions , cgamp can also be packaged into viral particles and extracellular vesicles ( figure 1 ) .
these particles can deliver cgamp and trigger sting - dependent signaling in newly infected cells .
this represents yet another mechanism by which cgamp produced by an infected cell can be transferred and propagate innate immune signaling in neighboring cells . as cgas responds to many viruses , including retroviruses such as hiv , it would be expected that bystander activation caused by cgamp transmitted through gap junctions or cgamp packaged into viral particles could be a common immune strategy employed against a wide array of viruses .
furthermore , cgas also produces cgamp upon sensing of bacterial dna , such as during mycobacterium tuberculosis infection , and many bacteria produce their own cyclic dinucleotides that are also recognized by sting and potently trigger the irf3 signaling pathway .
one could envision that during bacterial infection , either cgamp or bacterial cyclic dinucleotides could be transmitted from infected cells to neighboring bystander cells via gap junctions , thus ensuring and amplifying antibacterial immune responses as well .
many viruses disrupt retinoic acid - inducible gene 1 ( rig - i)-like receptor and tlr - mediated signaling .
for example , the positive - sense rna virus , hepatitis c virus ( hcv ) , encodes a viral protease , ns3/4a , which cleaves the rig - i and melanoma differentiation - associated protein 5 ( mda-5 ) signaling adapter mitochondrial antiviral signaling protein ( mavs ) . whether or how the host is capable of mounting a robust type i ifn response against hcv , in the face of immune evasion , has been unclear .
recent studies have indeed implicated bystander activation in mediating an effective type i ifn response as a result of cell  cell communication between hcv - infected hepatocytes and uninfected plasmacytoid dendritic cells ( pdcs ) . by taking advantage of the inability of pdcs to support viral replication
, it was shown through in vitro co - culture experiments that pdcs , not the infected hepatocytes , produced type i ifns in response to infection .
further investigation revealed that hepatocyte - derived exosomes containing viral rna were taken up by neighboring dcs , thereby triggering nucleic acid sensors in the neighboring pdcs ( figure 1 ) .
these exosomes are also detectable in the serum of hcv - infected patients , suggesting that they also mediate bystander activation during the natural course of hcv infection .
similarly , pdcs were activated in a tlr7-dependent manner , as a result of exosomal rna released from hepatocytes in an in vitro model of negative - strand rna lymphocytic choriomeningitis virus infection .
therefore , exosomal transfer of viral pamps to bystander immune cells may be a general mechanism employed by the immune system to circumvent viral inhibition of innate immune signaling .
this theme of viral nucleic acid transfer between host cells is not unique to mammals .
viral nucleic acid transfer to bystander cells , although exosome - independent , has been described in a drosophila melanogaster model of sindbis virus infection . in this case , infected host cells released unpackaged , viral double - stranded rna ( dsrna ) .
these nucleic acids were then taken up by uninfected cells , where they led to the induction of protective antiviral immunity throughout the fly via rna interference mechanisms .
mutant flies deficient in the dsrna uptake pathway were profoundly susceptible to infection with both drosophila c virus and sindbis virus .
thus , antiviral immunity in drosophila relies on cell  cell propagation of innate immune signals as well .
in addition to bystander responses to viral nucleic acids , there is also evidence that bystander responses triggered by other viral pamps are critical for orchestrating both innate and adaptive responses .
many pathogens , including influenza virus , would be expected to disarm the function of dcs , a critical cell type that serves as a bridge between innate and adaptive immunity . during influenza infection ,
the nlr family pyrin domain containing 3 ( nlrp3 ) is activated in response to the influenza m2 ion channel , leading to the formation of a multiprotein complex termed the inflammasome .
the nlrp3 inflammasome then activates the host protease caspase-1 , leading to processing and release of il-1 family cytokines , including il-1 and il-1. subsequently , it was found that il-1 receptor ( il-1r ) signaling , rather than direct sensing of viral infection by the prrs tlr7 or rig - i , was required for dc activation during influenza infection ( figure 1 ) .
il-1r signaling was both sufficient and necessary for dcs to upregulate expression of costimulatory molecules such as cd86 , migrate to lymph nodes and prime a virus - specific cd8 t - cell response .
why direct sensing of influenza virus by the prrs tlr7 and rig - i is not sufficient is unclear , but one possibility is that influenza , like many viral pathogens , may disarm signaling downstream of these innate immune sensors in directly infected dcs . given that many viral infections induce inflammasome activation , release of il-1 cytokines by infected cells and subsequent il-1 signaling to uninfected dcs may provide a failsafe mechanism for ensuring a successful immune response to a wide variety of viruses .
much like the gap junction - dependent transfer of cgamp during vaccinia infection , infected host cells are capable of using gap junctions to directly transfer pathogen recognition signals to uninfected bystander cells in response to bacterial stimuli as well . in response to treatment with the bacterial pamp lipopolysaccharide ( lps ) ,
immune cells and epithelial cells modulate expression of the gap junction protein connexin 43 ( cx43 ) .
furthermore , lps - mediated activation of dcs requires cx43-dependent gap junctions , suggesting that lps signaling triggers molecular signals of an unknown nature that are then propagated via gap junctions to maximize immune activation ( figure 1 ) .
these early studies pointed to a role for gap junction - mediated intercellular communication in host responses to bacterial stimuli .
subsequent studies described below have since shown that cell  cell communication mediated by gap junctions indeed do have a role in propagating the host response to bacterial pathogens . during infection with the extracellular gram - negative pathogen pseudomonas aeruginosa , tlr2
signaling induces a ca flux that stimulates nf-b and mapk signaling required for the recruitment and activation of neutrophils to the airway . in response to tlr2 ligands or p. aeruginosa infection , ca
fluxes could be transmitted from stimulated human airway cells to adjacent cells in a manner requiring cx43-mediated gap junctions ; this led to the increased production of the neutrophil - attracting chemokine cxcl8 by epithelial cells .
furthermore , treatment of p. aeruginosa - infected mice with pharmacological gap junction inhibitors significantly decreased neutrophil recruitment .
these findings suggest that the gap junction - mediated activation of neighboring bystander cells is critical for promoting antibacterial host defense in vivo .
the intracellular gram - negative pathogen shigella flexneri uses a type iii secretion system to translocate multiple bacterial effector proteins into the host cell cytosol to impair host cell signaling and production of cytokines and chemokines , such as the neutrophil chemoattractant il-8 .
for example , the effector ospg inhibits host ubiquitylation , thus blocking ib degradation and subsequent nf-b activation , whereas ospf is a phosphothreonine lyase that irreversibly dephosphorylates p38 and erk mapks . by employing microscopic analysis of s. flexneri - infected host cell monolayers at the single - cell level ,
it was found that infected cells were poor producers of il-8 . instead , within minutes post infection , infected cells potentiated nf-b , jnk and erk signaling in neighboring uninfected cells , leading to il-8 production by these bystander cells .
bystander il-8 production was also observed during infection with the bacterial pathogens listeria monocytogenes and salmonella enterica serovar typhimurium .
the induction of this bystander response required sensing of peptidoglycan - derived fragments by the intracellular receptor nucleotide - binding oligomerization domain - containing protein 1 ( nod1 ) in the infected cell , and then propagation of a signal to the neighboring cell via cell  cell contact through gap junctions .
the nature of this transmitted signal is unknown , but could involve secondary messengers such as calcium or other host - derived signals .
bystander activation in response to bacterial pamps or infection can also be mediated by soluble signals .
it was found that in response to lps , as well as viral pamps , a small number of early responder cells secrete ifn , which activates expression of antimicrobial genes in other cells to yield an efficient response at the population level .
uncovering this response required single - cell analysis , either through fluorescence microscopic analysis of rna expression or single - cell rna - sequencing in conjunction with various techniques to chemically or physically block cell  cell communication . a study investigating the immune response mounted against l. monocytogenes infection employed single cell approaches , involving fluorescence microscopy and flow cytometric analysis of epithelial cells infected by gfp - expressing l. monocytogenes .
directly infected cells were impaired in their ability to produce the chemokines cxcl2 and cxcl5 , suggesting that l. monocytogenes inhibits chemokine production . instead , these chemokines were primarily produced in neighboring non - infected epithelial cells , and this cell  cell communication was independent of gap junctions or cytokine secretion . instead , reactive oxygen intermediates , produced by nadph oxidase ( nox ) 4 in infected cells , mediated paracrine activation of neighboring bystander cells .
how nox4 is activated in response to l. monocytogenes infection is not entirely clear , but appears to involve detection of l. monocytogenes - derived ligands by nod2 and other cytosolic innate immune receptors . in another example of bystander activation
, a limited repertoire of soluble inflammatory cytokines that is selectively synthesized by infected cells can activate bystander cells to produce cytokines and subsequently amplify and diversify the immune response .
legionella pneumophila utilizes a type iv secretion system ( t4ss ) to translocate bacterial effector proteins that aid in establishing a membrane - bound organelle that supports intracellular infection .
this t4ss translocates several effectors , including lgt1 , lgt2 , lgt3 , sidi , sidl , pkn5 and lpg1489 , which potently inhibit host protein synthesis , in part , by impairing host translational elongation .
furthermore , l. pneumophila infection further inhibits host protein synthesis by suppressing translational initiation . despite a > 95% decrease in de novo protein synthesis ,
l. pneumophila - infected macrophages produce a robust cytokine response that paradoxically requires immune sensing of t4ss activity . a t4ss - dependent inflammatory response characterized by robust pro - inflammatory cytokine production and neutrophil recruitment to the lung
is observed in mice during pulmonary infection as well . to investigate how pro - inflammatory cytokines are made despite the effector - driven block in host translation ,
two independent studies used flow cytometry to distinguish infected from uninfected macrophages within the same population . in these studies ,
intracellular cytokine staining was performed following infection of macrophages with l. pneumophila reporter strains expressing either gfp or a t4ss effector translationally fused to the enzyme -lactamase , which modifies a fluorescent dye loaded into host cells following t4ss - mediated translocation . by tracking directly infected macrophages , it was found that these cells still synthesized the cytokines il-1 and il-1 de novo .
selective translation of il-1 and il-1 in infected macrophages required signaling through the tlr adapter myd88 through an as - yet - unidentified molecular mechanism . 

legionella - infected cells then go on to process and secrete mature il-1 cytokines upon cytosolic sensing of bacterial ligands such as flagellin and lps , and subsequent inflammasome activation .
thus , inflammasome - dependent cytokines escape the effector - driven translational block , as they are still selectively translated and released by l. pneumophila - infected cells . the translational block in l. pneumophila - infected macrophages
, however , did hinder their ability to produce other important pro - inflammatory proteins , such as il-6 , il-12 , tnf and the costimulatory molecule cd86 .
analysis of pulmonary infection similarly found that directly infected alveolar macrophages were also impaired in their ability to produce tnf , but still upregulated expression of il-1 and il-1. tnf was produced primarily by uninfected alveolar macrophages , as well as a variety of other bystander immune cell types , including neutrophils , inflammatory monocytes and dcs .
pro - inflammatory responses by these bystander populations required il-1 signaling , as mice lacking the il-1r exhibited a marked reduction in tnf and il-12 production , and cd86 expression by bystander cells in vivo ( figure 1 ) .
how il-1r signaling activates bystander cells during in vivo infection is not yet clear , in part , because it is unknown whether cell - intrinsic il-1r signaling is required for bystander immune responses to l. pneumophila .
interestingly , il-1 signaling was neither required nor sufficient to elicit expression of tnf and il-6 in bone marrow - derived macrophages ( bmdms ) in vitro , suggesting a role for other selectively translated signals in mediating bystander activation in bmdms and potentially during pulmonary infection . because inflammasomes respond to many bacterial infections
, it would be expected that in addition to the key role of il-1 signaling in driving neutrophil recruitment , il-1 would similarly drive bystander production of pro - inflammatory cytokines in response to other bacterial pathogens as well .
the cytokine il-12 has a major role in controlling many intracellular bacterial pathogens , including mycobacterium spp .
infecting yet40 reporter mice expressing the il12p40 gene linked via an internal ribosome entry site to yfp with mycobacterium bovis bacillus calmette - gurin ( bcg ) expressing dsred fluorescent protein enabled tracking of both the cellular source of il-12p40 and the location of bcg bacteria .
dcs were the major source of il-12p40 , and these cells were not only uninfected , but also did not co - localize with bcg .
further analysis indicated that dcs directly infected with bcg exhibited impaired il-12p40 production , and that soluble mycobacterial components could initiate the il-12p40 production in uninfected dcs during in vitro infection .
interestingly , macrophages infected with bcg or m. tuberculosis release exosomes containing bacterial lipoproteins and lipoaraminomannan , and these exosomes stimulate the cytokine production in naive macrophages ( figure 1 ) .
furthermore , exosomes isolated from the balf and serum of bcg - infected mice can induce macrophages to produce cytokines .
in addition to exosomes , m. tuberculosis - infected macrophages actively release bacterial membrane vesicles containing bacterial lipoglycans and lipoproteins that induce tlr2 signaling and cytokine responses in uninfected macrophages during in vitro infection . collectively , these studies suggest that bystander activation occurs during infection with various mycobacterium spp . , and that release of bacterial ligands by infected cells may provide another possible mechanism by which bacterial infection can be detected by uninfected bystanders .
in addition to cell  cell communication via cytokines or bacterial pamps , there is evidence that infected cells release innate immune signaling machinery that can be taken up by bystander cells .
these preformed inflammasome complexes were internalized and induced il-1 maturation in neighboring bystander cells , without the need for additional inflammasome - triggering stimuli ( figure 1 ) .
this mechanism may be active in vivo , as extracellular inflammasome complexes were visualized in the lymph nodes of mice infected with p. aeruginosa , and injection of purified inflammasome complexes into mice led to il-1-dependent recruitment of neutrophils .
thus , release of inflammasome complexes by infected cells represents yet another mechanism for propagating intercellular communication between infected and bystander cells .
there is evidence that bystander innate immune activation occurs in response to intracellular protozoan parasites as well .
macrophages infected with leishmania amazonensis released extracellular vesicles that enhanced the production of pro - inflammatory cytokines , such as il-12 and tnf , by uninfected macrophages in vitro ( figure 1 ) .
these extracellular vesicles appeared to be of host origin , but the nature of the immunostimulatory ligands contained within the vesicles and whether they are of host or parasite origin is unclear .
extracellular vesicles isolated from the plasma of plasmodium berghei - infected mice or from plasmodium falciparum - infected rbcs could also activate nave host cells in vitro .
the extracellular vesicles released by p. falciparum - infected red blood cells ( rbcs ) were of host origin , and contained both host proteins and membrane - associated parasite antigens . during in vivo toxoplasma gondii infection of mice
how these bystander cells produce il-12 in response to t. gondii is unknown , but presumably involves immune sensing of a soluble host or parasite factor . as the host immune system evolves bystander activation mechanisms to deal with a number of immune attenuation approaches by pathogenic microbes , one might suspect that pathogens , in turn , would evolve to overcome bystander activation  a further manifestation of the red queen hypothesis .
indeed , using t. gondii expressing an effector : cre recombinase fusion protein that can be translocated into host cells , in conjunction with reporter mice that express the fluorescent protein zsgreen following cre - mediated recombination , it was revealed that t. gondii injects its effector proteins into both infected and uninfected cells .
effector injection into both infected and uninfected cells has biological consequences , as injection of the rhoptry protein , rop16 , leads to robust stat6 activation in both infected and uninfected cells .
stat6 activation by t. gondii is thought to inhibit il-12 production , a cytokine essential to parasite resistance .
this ability to inject effectors into both infected and uninfected cells may critically enable t. gondii to attenuate innate immune activation in bystander cells .
thus , by injecting effectors into uninfected , as well as infected cells , the parasite can manipulate the host cytokine response to enable the parasite 's survival .
lewis carroll wrote in his 1871 novel , through the looking - glass , from which the red queen hypothesis was derived ,  now , here , you see , it takes all the running you can do , to keep in the same place .
if you want to get somewhere else , you must run at least twice as fast as that ! '
the observation that populations of hosts and pathogens are constantly under selective pressure to maintain a competitive advantage over one another embodies this idea . as pathogens have evolved numerous mechanisms to maintain intracellular niches within their hosts , host cells , in turn , have evolved ways to alert the immune system of the presence of an infection . over time , host cells have developed means to communicate with neighboring , uninfected bystander cells in situations in which primary immune activation mechanisms have been silenced in the directly infected cells .
this review synthesizes some of the most recent literature , regarding bystander activation across a diverse pathogenic microbial taxonomy , as it is important to the fields of microbial pathogenesis and immunology to understand some of these mechanisms and how they function to initiate and shape a variety of immune responses . as researchers continue to elucidate the complexities of the host 
microbial relationship , we posit that bystander activation will be shown to be a critical component in many of these dynamics .
we expect that there will be diverse mechanisms driving bystander activation , and that many pathogens will evade or manipulate mechanisms of bystander activation for their own advantage .
understanding the role for uninfected bystander cells in infectious pathologies is not only important for the advancement of our understanding of host : pathogen biology , but it also will continue to drive the forefronts of medicine , evolutionary biology and the vast study of infectious diseases .
 originally described by the late evolutionary biologist leigh van valen , the red queen hypothesis posits that the evolutionary arms race between hosts and their pathogens selects for discrete , genetically encoded events that lead to competitive advantages over the other species . 
 examples of immune evasion strategies are seen throughout the co - evolution of the mammalian immune system and pathogens , such as the enzymatic inactivation of nuclear factor-b signaling or host translation by pathogen - encoded virulence factors . 
 such immunoevasive maneuvers would be expected to select for the evolution of innate immune counterstrategies . 
 recent advances in our understanding of host immunity and microbial pathogenesis have provided insight into a particular innate immune adaptation , termed bystander activation . 
 bystander activation occurs as a consequence of infected cells alerting and instructing neighboring uninfected cells to produce inflammatory mediators , either through direct cell contact or paracrine signals . 
 thus , bystander activation can allow the immune system to overcome the ability of pathogens to disarm immune signaling in directly infected cells . 
 this review presents an overview of the general hallmarks of bystander activation and their emerging role in innate immunity to intracellular pathogens , as well as examples of recent mechanistic discoveries relating to the bystander activation during infection with specific pathogens relevant to human health and disease . 
Introduction
Viral pathogens
Bacterial pathogens
Protozoan pathogens
Conclusion