Discussion
Gene losses in inflammation–related gene families and positive
selection in single–copy genes associated with immune and cell repair
functions in mammalian models have been evident since the very first bat
genome assemblies were published (G. Zhang et al., 2013). Although
subsequent studies have confirmed those initial results (Ahn et al.,
2016; Seim et al., 2013), confidence in assessing both gene losses and
gene family expansions has strengthened only recently, with the
publication of highly contiguous assemblies for a few bat species (Jebb
et al., 2020; Scheben et al., 2020). Examining a comprehensive sample of
bat lineages while checking against high quality genome assemblies and
multi organ RNA Seq, our analyses reveal system wide gene losses with
the potential to modify the sensitivity, targets, and magnitude of
immune responses across all bats. These inferred losses are particularly
concentrated along inflammasome activation pathways, which are triggered
by the innate immune recognition of pathogenic signals through both
pathogen-associated molecular patterns (PAMPs) and damage associated
molecular patterns (DAMPs). In contrast with more pathogen–driven
PAMPs, DAMPs result from host cellular distress signals such as
mitochondrial stress and reactive oxygen species (ROS) (Zheng, Liwinski,
& Elinav, 2020), which bats produce during active flight (Costantini,
Lindecke, Petersons, & Voigt, 2019). Bat cells, in turn, display
exceptional mechanisms of repair (Pickering, Lehr, Kohler, Han, &
Miller, 2014) and resist damage (Harper, Salmon, Leiser, Galecki, &
Miller, 2007), connecting molecular signaling and cell processes to
extreme longevity (Salmon et al., 2009; Wilkinson & Adams, 2019).
Based on our genomic surveys, immune-related losses can be divided into
three categories: the epithelial defense receptors (defensins), the
Natural Killer gene complex (NKC) and the interferon-induced pathway
(IFI; HIN; PYHIN) (Figure 2). This particular combination of losses in
crucial components of immune activation seems surprising, as it would
imply that bats mount a low intensity immune response to primarily
intracellular pathogens: viruses. But integrating these genomic findings
with published functional data suggests complex, systemic adaptation, in
line with both previous analyses of bat immune system responses (A.
Banerjee et al., 2020; Basler, 2020; P. Zhou, 2020) and the growing body
of evidence for cellular mechanisms underlying longevity (Z. Huang,
Whelan, Dechmann, & Teeling, 2020; Z. Huang et al., 2019, Kacpryzk et
al., 2017). We review these losses in a stratigraphic order, from the
outer cellular matrix to the inner cellular pathways, starting with the
defensins.
While defensins are the primary barrier of the immune system, with broad
antimicrobial activity that covers bacteria, fungi and viruses (Semple
& Dorin, 2012; Xu & Lu, 2020), bat defensin losses consist mainly of
orthologs of genes localized to epithelial cells. Defensins can function
as modulators of the host’s cell surface receptors, and α and β
defensins genes have pleiotropic effects on the regulation of
carcinogenesis and inflammation (Xu & Lu, 2020). By acting as
chemokines to alter the adaptive immune response, defensins also serve
as a bridge between innate and adaptive immunity (Grigat, Soruri,
Forssmann, Riggert, & Zwirner, 2007). In bats, both α and β defensin
genes have undergone a rapid evolutionary change through either loss or
positive selection (Table 1, Figure 2a, Supplementary Table 4). In
humans, defensins can elicit proinflammatory cytokine production
(Niyonsaba et al., 2010; Wiens, Wilson, Lucero, & Smith, 2014), but
overexpression of certain defensins can actually enhance viral infection
(Rapista et al., 2011). We hypothesize that specific defensin losses in
bats (Figure 2a) complement several other mechanisms (Ahn et al., 2019;
A. Banerjee et al., 2017; Xie et al., 2018) contributing to a dampened
inflammatory response, reduced host–driven damage from viral
infections, and enhanced longevity (Baker & Schountz, 2018; Brook &
Dobson, 2015; Gorbunova et al., 2020). For example, modifying defensin
repertoires on epithelial cells would result in fewer instances of both
immune cell recruitment and initiation of inflammatory pathways known to
damage healthy tissue (e.g., focal necrosis in lungs, spleen and lymph
nodes during the inflammatory response during SARS-Cov2 infection (Merad
& Martin, 2020)). In humans, loss of β-defensins prevents the
inhibition of neutrophil apoptosis and thus averts the production of
proinflammatory cytokines and chemokines (Nagaoka, Niyonsaba,
Tsutsumi-Ishii, Tamura, & Hirata, 2008), avoiding the amplification of
the immune response, and may have a similar effect in bats. Losses of
some epithelial surface defensins would thus reduce inflammation without
compromising responses to intracellular pathogens.
Another result with inferred implications for reducing proinflammatory
reactions involves losses of Natural Killer (NK) receptors that play an
important role in the recognition of MHC-I molecules and regulation of
cytotoxic activity against virus–infected cells. While killer-cell
immunoglobulin like receptors (KIR) and killer cell lectin-like
receptors (KLR) receptor losses has been previously reported forPteropus alecto and Myotis davidii (Papenfuss et al.,
2012; G. Zhang et al., 2013), our analyses confirm these losses across
Chiroptera (Supplementary Table 4). Although the Killer Cell
Lectin Like Receptor K1 (KLRK1 or NKG2D) gene is present in bats, its
ligands, gene subfamilies RAET1 and H60 responsible for
binding and activating NKG2D receptors, recruiting natural killer cells,
and stimulating them to secrete Interferon gamma (IFN-γ) (Zhi et al.,
2010), were absent in all bat species (Figure 2b).
We hypothesize that these losses lead to low recruitment of
proinflammatory NK cells and reduce B-cell signaling (Arapović et al.,
2009; Stolberg et al., 2014; Takada et al., 2008; Wortham et al., 2012),
as they do in mice and humans. Loss of this particular mechanism of
activation of the MHC-I pathway prevents proliferation of immune cells,
which can be cytotoxic, proinflammatory, and targets of viral infections
(Djelloul, Popa, Pelletier, Raguénez, & Boucraut, 2016; Wortham et al.,
2012). For example, NKG2D–deficient mice infected with influenza
viruses exhibit less airway damage and reduced inflammation without
compromising viral clearance; NKG2D stimulation is a central pathway to
viral-mediated NK cell hyperresponsiveness (Wortham et al. 2012), and
has been shown to be involved in age-dependent COVID–19 severity (Akbar
& Gilroy, 2020). During viral exposure, rarer activation of NKG2D
function would therefore lead to less inflammatory exacerbation.
Reducing instances of NKG2D activation might also reduce B cell
signaling, as it occurs in NKG2D–deficient mice (Lenartić et al., 2017;
Zafirova et al., 2009), and complements losses of immunoglobulin heavy
chain variable regions IGHV1, IGVH3 and IGHV14 genes that modify the B
cell receptor signaling pathway, and thus B lymphocyte differentiation
(M. Banerjee, Mehr, Belelovsky, Spencer, & Dunn-Walters, 2002;
McHeyzer-Williams, Okitsu, Wang, & McHeyzer-Williams, 2012; Reddy et
al., 2010). Based on the roles of both NKG2D and B cell activation in
promoting inflammation in viral infection, and since some viral proteins
have been shown to specifically target the NKG2D receptor via the RAET1
and H60 loci (Arapović et al., 2009), we propose that these losses
resulted from selection during viral infections early in the
evolutionary history of bats. While the functional implications for bats
need to be tested, in humans, lack of specificity of the T and B cells
in children results in a broader immune response to novel viruses
(Pierce et al., 2020), and it may confer analogous advantages in bats.
Complementing losses in defensins and NK signaling, the third large
group of gene losses involves the IFN-γ pathway (Figure 2c). While
representatives of the PYRIN and HIN domain (PYHIN) gene family, immune
sensors of cytosolic DNA activating the inflammasome and IFN-γ, are
present in all mammals, they have not been found in any of the bat
genomes analyzed thus far examined (Ahn et al., 2016; G. Zhang et al.,
2013; Jebb et al., 2020). Previous genomic analyses linked losses in
this inflammasome pathway to the unique demands of bat flight and in
response to increased ROS production (G. Zhang et al., 2013), but there
are immune implications as well. In other mammals, the presence of
dsDNA, DAMPs and PAMPs, or, especially, bacteria and DNA viruses,
induces the (PYHIN) AIM2 inflammasome, while the IFI16 inflammasome
(Interferon-inducible protein 16, also missing in bats) recognizes
viruses replicating in the nucleus (Zheng et al., 2020). Hence, these
bat gene losses could undermine innate defense against viruses. We
hypothesize that bats have evolved mechanisms to overcome this potential
disadvantage in rapid recognition and response against viruses through
expansion of MHC-I class genes (Supplementary Table 7). These genes are
involved in the recognition and binding of intra cellular peptides, and
previous studies have described a unique 5–amino acid insertion at the
exon 2 peptide binding region (PBR) on bats which may allow the host to
recognize longer peptides (Ng et al., 2016; Papenfuss et al., 2012).
Besides implications for immunity, IFN-γ pathway gene losses also point
to changes in autophagy. In mice, loss of the IFN-γ inducible immunity
related GTPase gene (IRGM1 and IRGM2) results in an IFN-γ induced
autophagic death program in lymphocytes (Feng et al., 2008). Along with
the loss of other IFN-γ related genes (IGTO, IIGP, TGTP2), these losses
may help achieve apoptosis of infected cells without runaway
inflammation.
While some mechanisms of activation of IFN-λ are lost in bats, IFN-γ
itself is under positive selection within branches (Table 1
Supplementary Table 7). IFN-γ is a crucial part for the first line of
defense against viruses, helps shape adaptive immune memory (Schroder,
Hertzog, Ravasi, & Hume, 2004), and its deficiency increases
inflammation (Loo et al., 2017). Thus, evolutionary adaptation may have
shaped bats’ unique ability to induce a rapid antiviral response without
triggering runaway inflammation. This fine-tuned response may be
achieved by expressing high levels of IFN-γ early on, which recruits
broad-spectrum immune cells to the site of injury, while negatively
regulating the IFN-γ pathway receptors that trigger inflammation (Ahn et
al., 2019; Ferber et al., 1996).
In contrast to a pattern of proinflammatory signal losses, most other
variation in gene families in Chiroptera corresponded to cell processes
and metabolic functions. Shifts from the ancestral bat insectivorous
diet to including nectar and fruit and the resulting mutualistic
relationships between bats and plants appear to have led to elevated
rates of diversification and the evolution of new morphological traits
(Dumont et al., 2012; Jones, Bininda-Emonds, & Gittleman, 2005), but
gene family evolution has remained underexplored. Regarding significant
expansions (Supplementary Table 7), we identified few —only nine—
sets of duplications independently replicated across all pteropodids and
phyllostomids with convergent, plant–based diets (Figure 1). In
addition to a trace amine associated receptor (TAAR) of unknown
chemosensory function (Liberles & Buck, 2006) and a putative homolog of
the yeast protein transport protein YIP1, two genes stand out as
candidates for diet–linked adaptive gene family evolution: those
encoding homologs of inositol monophosphatase 1 (IMPA1) andintegrin alpha-D/beta-2 (ITAD). Glycolysis, the metabolic pathway
that breaks down glucose to ultimately phosphorylate more ADP into ATP
than the reverse, begins with the phosphorylation of glucose into
D-glucose 6-phosphate (Berg, Tymoczko, & Stryer, 2002). This
metabolite, however, cannot diffuse through the membrane and is thus
highly osmotic; its accumulation would cause cells to swell. Through the
synthesis of myo -inositol from D-glucose 6-phosphate, IMPA1
provides one avenue to protect cells, particularly in the brain
(Parthasarathy, Parthasarathy, & Vadnal, 1997), from the osmotic stress
of this glucose metabolite (Rafikov et al., 2019). We found independent
IMPA1 duplications in the pteropodid ancestor, A. jamaicensis ,A. caudifer , P. discolor , and the common ancestor of
phyllostomids and Mormoops . Except for the aerial insectivoreMormoops , all the lineages with IMPA1 duplications include nectar
and fruit in their diet (Figure 1), are expected to at least
occasionally experience high blood glucose levels (Amitai et al., 2010;
Ayala & Schondube, 2011; Kelm, Simon, Kuhlow, Voigh & Ristow, 2011;
Welch, Herrera & Suarez, 2008; Meng, Zhu, Huang, Irwin, & Zhang,
2016), and therefore require options for processing metabolites from
glycolysis. Although beta integrins, including ITAD, are regulators of
leukocyte function and therefore not annotated as directly involved in
metabolism, leukocyte adhesion has been found to modulate glucose
homeostasis via lipid metabolism (Meakin et al., 2015). Specifically,
mice deficient in a paralogous beta-2 integrin become spontaneously
obese in old age despite a normal diet (Z. Dong, Gutierrez-Ramos, Coxon,
Mayadas, & Wagner, 1997), and when fed a fat rich diet show obesity,
inflammation, high neutrophil activity and insulin resistance in
skeletal muscle (Meakin et al., 2015). Likewise, mice deficient in this
same integrin are unable to respond to fasting by increasing fat uptake
and reduce insulin levels slowly compared to normal mice (Babic et al.,
2004). We found single ITAD duplications in lineages that include sugar
rich foods in their diet: ancestral pteropodids and phyllostomids, as
well as Leptonycteris yerbabuenae , two each inMacroglossus , Anoura , and Tonatia , and three inArtibeus jamaicensis . While the function of these
lineage–specific bat paralogs remain unknown, their phylogenetic
distribution warrants future exploration and functional analysis.
In summary, our results, grounded on the most comprehensive survey of
bat genomes to date, suggest bats have evolved complex mechanisms of
inflammasome regulation. These may have evolved to prevent uncontrolled
inflammatory response against DAMPs byproducts of the high metabolic
rate required for powered flight (Banerjee et al., 2017; Banerjee et
al., 2020; Subudhi, Rapin & Misra, 2019; Xie et al., 2018), to better
respond against intra-cellular pathogens such as viruses, or some
combination of both. Regardless of the ecological origin of selection,
compared to mammals such as humans or mice, bat genomes reveal
systemwide immune evolution that prevents or dampens aggressive
inflammatory responses. In contrast with these gene losses, we found
significant expansions in gene families involved with glucose
degradation, coinciding with the transition from a diet based mainly on
insects to a high-glucose content diet that includes fruit and nectar.
By undertaking large-scale comparative genomic analyses encompassing
many ecologically divergent lineages, the present study demonstrates the
impact of genomics in non-model organisms. Such analyses allow
elucidating the broad evolutionary mechanisms in a given clade, with
potential for functional implications. Yet, heterogeneity in assembly
quality continues to limit the scope of inference. Hence, the need to
generate high quality genomes for future studies endures.