Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders often co-occurring in the same patient, a feature that suggests a common origin of the two diseases. Consistently, pathological inclusions of the same proteins as well as mutations in the same genes can be identified in both ALS/FTD. Although many studies have described several disrupted pathways within neurons, glial cells are also regarded as crucial pathogenetic contributors in ALS/FTD. Here, we focus our attention on astrocytes, a heterogenous population of glial cells that perform several functions for optimal central nervous system homeostasis. Firstly, we discuss how post-mortem material from ALS/FTD patients supports astrocyte dysfunction around three pillars: neuroinflammation, abnormal protein aggregation, and atrophy/degeneration. Furthermore, we summarize current attempts at monitoring astrocyte functions in living patients using either novel imaging strategies or soluble biomarkers. We then address how astrocyte pathology is recapitulated in animal and cellular models of ALS/FTD and how we used these models both to understand the molecular mechanisms driving glial dysfunction and as platforms for pre-clinical testing of therapeutics. Finally, we present the current clinical trials for ALS/FTD, restricting our discussion to treatments that modulate astrocyte functions, directly or indirectly. Amyotrophic Lateral Sclerosis (ALS) is the most common form of adult-onset motoneuron disease, with an incidence of 1–2 cases/year/100,000 individuals. The disease presents with a wide variety of symptoms arising from motoneuron degeneration. The site of symptom onset normally provides a first criterion to stratify patients. More specifically, bulbar onset is declared when the disease appears with difficulties in swallowing (dysphagia) and articulating speech (dysarthria), while fasciculation, tremors, and muscular tone loss are hallmarks of spinal-onset ALS. In addition to these motor disabilities, about 15% of patients develop cognitive and behavioral impairments that call for an additional diagnosis of Frontotemporal Dementia (FTD). Patients can be further stratified on the basis of the etiological origin of the disorder. In 90–95% of occurrences, it appears sporadically (sALS), with many environmental and behavioral agents implicated in its pathogenesis, whereas it is inherited in the remaining 5–10% of instances, mainly as an autosomal dominant character (fALS). Mutations in several genes have been associated with the disease. From a neuropathological standpoint, post-mortem analysis reveals the loss of the motor cortex and spinal cord motor neurons as well as reactive gliosis. In addition, the cytoplasm of surviving neurons and glial cells shows abnormal proteinaceous aggregates, known as inclusions, whose defining element is the protein Transactive Response DNA-binding Protein 43 (TDP-43), which is present in 95% of cases . Hereafter, we will, therefore, refer to FTD when discussing findings relative to the clinical condition, and FTLD will be reserved to evidence gathered at the biochemical, pathological, and cellular levels. Importantly, about 20% of FTD individuals develop motor dysfunction, meeting the criteria for an additional ALS diagnosis. The majority of cases cannot be ascribed to a known cause, while about 30% of occurrences are of a genetic origin, as mutations in different genes, including some linked to fALS, have been associated with the disease (L)-deprenyl-D2, which enables the measurement of monoamine oxidase B (MAO-B) levels, has indeed served as a powerful tool to track astrocyte activation by positron emission tomography (PET) in ALS . ComplemA complementary approach to monitor astrocyte impairment along disease progression would be to identify and validate soluble biomarkers that can report such dysfunction. For example, assessing the BBB integrity may serve as a proxy to monitor astrocyte activity since these cells play a crucial role in the formation and maintenance of the barrier . InteresThese conflicting results urged for the identification and validation of alternative astrocyte-specific biomarkers, as BBB dysfunction may also arise from impaired functionality of other cellular partners contributing to its homeostasis. To reach this goal, several studies were performed to investigate the levels of proteins, typically expressed by activated astrocytes, in the cerebrospinal fluid of patients affected by different neurodegenerative diseases. Interestingly, chitinase-3-like protein 1  expression was reported to discriminate patients who received a double ALS/FTD diagnosis from those with FTD with no impairment of motor capabilities . MoreoveAnother glial protein, which is currently under investigation as a potential biomarker, is the cytoskeletal protein GFAP. In serum, its concentration is higher in ALS patients with cognitive impairment  and negaThese biomarker studies point towards the intriguing possibility that the dysfunction of astrocyte subpopulations could be the ultimate disease modifier, aggravating neurodegeneration in different CNS regions and, in turn, leading to the development of more complex disease manifestations. Further indirect evidence supports this hypothesis: the composition of the gut microbiota, which has an important tuning role on astrocytes homeostasis ,70,71,72C9orf72 gene plays a role in subduing a pathological inflammatory response triggered by gut bacteria ](2-)]-copper (CuATSM) reduced elevated basal and ATP-linked mitochondrial respiration and increased glycolysis, leading to enhanced lactate release in human astrocytes derived from sALS patients or fALS cases carrying different genetic lesions. However, the molecular mechanisms underlying these events have not been fully elucidated and require further attention [The discovery that ALS/FTD astrocytes display metabolic impairment led to the hypothesis that motor neuron toxicity could arise from a lack of adequate sustenance. In agreement with this vision, two strategies proved to be informative. As mouse ALS astrocytes display defective lactate release, a supplementation of mouse ALS astrocyte-motor neuron co-culture medium with this metabolite was first shown to rescue neuronal demise . A simil lactate . Consistttention . Interesttention ,158.G93A downregulate miRNA146a. Restoring its expression in astrocytes reverted their neurotoxic phenotype [More recently, microRNAs were identified as another class of potential mediators of astrocyte-mediated neurotoxicity. In particular, mouse astrocytes expressing hSOD1henotype ,159. Inthenotype . As re-ehenotype . Noteworhenotype .C9orf72 HRE as well as in the spinal cord of sALS cases [PTEN) [Another astrocyte-derived microRNA of interest is miR-494-3p. This small RNA is released by astrocytes in the exosomes, and its expression is reduced in cells differentiated from patients carrying LS cases . RestoriLS cases . Furthers [PTEN) , an evens [PTEN)  and mitis [PTEN) . It is, G93A expressing astrocytes. The results were then submitted to a novel bioinformatics analysis along with pre-existing data describing the transcriptome of motoneurons in order to identify and prioritize noxious ligand-receptor interactions. This strategy led to the discovery of aberrant signaling originating from the release of the amyloid precursor protein (APP) from astrocytes and the activation of the death receptor-6 (DR6) on motoneurons [Taken together, these studies suggest that the toxic interplay between astrocytes and neurons in ALS/ FTLD is likely mediated by a combination of several different aberrant cellular pathways in both cell types. This consideration implies that efforts should be made to prioritize the most robust candidates in the quest to identify the toxic element of the deadly astrocyte-neuron interplay in ALS. To this end, Mishra et al.  took an oneurons . Importaoneurons .SOD1. More specifically, a conditioned medium from ALS-astrocytes was reported to induce a robust inflammatory response in a microglia-like cell line [The cross-talk between different populations of glial cells has received less attention than the cell–cell communication signaling occurring between astrocytes and neurons. The characterization of transgenic animals with selective TDP-43 depletion from astrocytes, however, led to the discovery that astrocytes themselves acquire a neuroinflammatory phenotype, and microglia increase their reactivity markers . These mell line . This miell line . This evGRN mutations suggested that diseased astrocytes display enhanced chemokine (C-X-C motif) ligand 1 signaling towards vascular cells, a cascade known to be associated with augmented BBB permeability [The BBB and the blood–spinal cord barrier are the structures that tightly regulate the exchange of substances between the bloodstream and the CNS parenchyma. Perivascular astrocytes locate their endfeet around the capillaries and establish functional interactions with components of the BBB. They regulate its functions by exchanging signals with the other cell constituents, namely, endothelial cells and pericytes . Notablyeability . Finallyeability ,184,185 eability .G93A mice [Several lines of evidence indicate that astrocyte dysfunction is not only a consistent feature of the ALS/FTLD spectrum but also contributes to the establishment of a neurotoxic environment through aberrant interactions with neurons and other neural cell types. Thus, astrocytes may represent an attractive target for therapeutic intervention in ALS/FTD. The previously mentioned transplantation experiments in animal models ,88,89 al93A mice .Neural progenitor cells engineered to differentiate to astrocytes and to secrete GDNF (CNS10-NPC-GDNF) were produced under good manufacturing practice for a phase I/IIa clinical trial (NCT02943850) to test the safety of unilateral spinal cord injection in a small cohort of ALS patients . The stuSOD1. The success of these and several other studies aimed at silencing SOD1 in various CNS populations  led to was safe , a subsewas safe . NotablyCHMP2B variants rather increase glutamate uptake and metabolism [Among the different mechanisms mediating astrocyte toxicity towards neurons, functional impairment of the astrocyte plasmalemmal glutamate transporter EAAT2 has gained considerable interest as a potential therapeutic target from the milestone discovery that the antibiotic ceftriaxone enhances its expression and ameliorates the phenotype in mouse models of ALS  and FTLDtabolism  suggestsAnother signaling pathway that raised considerable interest as a potential therapeutic target within astrocytes is the antioxidant response mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) . The ratIn the previous section, we discussed how restoring the metabolic balance in ALS/FTD astrocytes might halt neuronal death. Intriguingly, the results of a pilot clinical trial (NCT02288091) showing the safety of inosine supplementation to ALS patients have been recently published . AlthougIn this review, we have extensively discussed considerable evidence demonstrating that astrocytes play a major role in the pathogenesis of ALS and FTLD. These observations suggest that any therapeutic regimen designed to halt neurodegeneration should include agents rescuing glial dysfunctions. Along with pharmacological interventions, rehabilitative training should be also considered to favor morphological remodeling and to improve the functional performance of astrocytes. Physical exercise ,226,227


Genetic variants are traditionally known to shape the susceptibility to neuropsychiatric disorders. An increasing number of studies indicate that remodeling of the gut microbiome by genetic variance serves as a versatile regulator of gut-brain crosstalk and behavior. Evidence also emerges that certain behavioral symptoms are specifically attributed to gut microbial remodeling and gut-to-brain signals, which necessitates rethinking of neuropsychiatric disease etiology and treatment from a systems perspective of reciprocal gene-microbe interactions. Here, we present an emerging picture of how gut microbes and host genetics interactively shape complex psychiatric phenotypes. We illustrate the growing understanding of how the gut microbiome is shaped by genetic changes and its connection to behavioral outcome. We also discuss working strategies and open questions in translating associative gene-microbiome-behavior findings into causal links and novel targets for neurobehavioral disorders. Dual targeting of the genetic and microbial factors may expand the space of drug discovery for neuropsychiatric diseases. Althoug20. Interestingly, gut dysbiosis has been observed as a common trait for neuropsychiatric disorders, and emerging data are suggesting that host genetics and the gut microbiome interdependently regulate different complex behaviors in genetic neurological disorders21. Therefore, it is reasonable to propose that dissecting the mechanisms through which host and microbial factors regulate complex behaviors will not only expand our understanding of neurobehavioral disorders but may also broaden the way of novel therapies. Here, we present a growing picture from animal studies that gut microbe interaction with host genetics to interactively shape complex psychiatric phenotypes. We discuss the challenges and potential strategies in gaining mechanistic insights into the genetic-microbe interplay in behavior control, with a major focus on microbial metabolites as signaling molecules for drug discovery. We also envision how advances in this frontier may potentially change the way we view and treat these complex mental illnesses in the clinic.The gut microbiome can modulate host behaviors in a very powerful manner via the gut-brain axis22. For example, a spectrum of gastrointestinal disorders, such as constipation, diarrhea, abdominal pain, vomiting, and poor nutritional absorption, have been reported in up to 90% of children with ASD23. Likely, MDD patients largely exhibit abnormality in gut motility, dyspepsia, and increased susceptibility to colitis. Also, patients with BD exhibit an increased frequency of gastrointestinal illnesses, such as inflammatory bowel disease, which has been mechanistically connected to microbial community function24. Gut dysfunction and associated metabolic and immune changes could in turn aggravate the behavioral symptoms, such as anxiety and social disruption25, thus forming a vicious cycle that calls for multi-dimensional intervention.In addition to brain-centric symptoms, somatic complications have emerged as a common clinical problem for neuropsychiatric diseases. Gut dysfunction, among others, seems to be the most common comorbidity with the behavioral changes26. Not surprisingly, gut microbial remodeling has now been extensively observed in patients with neuropsychiatric disorders , a progressive neurological disorder characterized by autism-like behaviors, is thought to be caused by mutations in the Mecp2 gene31. Recent study has moved the field one step further by showing that RTT patients harbored a gut microbiome featuring reduced microbial richness and a dominance of Bifidobacterium, Actinomyces, Lactobacillus and Enterococcus32. In addition, ample clinical findings show that the gut microbiome is altered in MDD patients33.Changes in gut physiology are well known to affect the colonization and fitness of the commensal microbial community. In addition, altered feeding behavior and nutritional status, commonly observed in neuropsychiatric diseases, are powerful in shaping the gut microbial configurationisorders . For exaChd8± mice revealed changes in gut microbial structure and amino acid transporters, which were linked to an increased glutamate/γ-aminobutyric acid (GABA) ratio in the brain34. Similarly, a previous study in Shank3KO mice reported a significantly different gastrointestinal morphology and altered microbial composition41. A recent study in RTT mice also reported alteration of gut microbiome across postnatal development as behavioral symptoms appear and progress42. Together, the extensive observation of gut microbial remodeling in clinical patients and preclinical models sheds an additional layer of insights into neuropsychiatric diseases and genetic underpinnings  mice showed that ASD microbiota was sufficient to induce hallmark autistic behaviors, accompanied by discrete metabolome profiles50. Intriguingly, treatment of the BTBR ASD mouse model with key differential microbial metabolites improved behavioral abnormalities and modulated neuronal excitability–inhibition balance in the brain, providing further causal evidence for microbial factors. Interestingly, treatment with Lactobacillus reuteri (L. reuteri) was shown to generally rescue social deficits in Shank3bKO and other environmental and idiopathic ASD models35. Also, of interest is the finding that L. reuteri6475 intervention selectively rescued the social deficits but not the hyperactivity phenotype in Cntnap2KO mice by bolstering the oxytocinergic system21. Together, these studies provide strong evidence that the gut ecosystem plays an important regulatory role in many aspects of ASD symptomatology.ASD is traditionally thought to be a neurological disease with a high heritability. Studies in 2013 have shown direct evidence that gut microbiota could modulate autism-related behavior2. However, the heritability estimates for MDD (40%) are lower than those for other neuropsychiatric illnesses (between 75% and 80%), implying that additional factors are involved. Several preclinical studies have shown that genetic manipulation can effectively modulate depression-like behavior, with the mechanism being partially attributed to the gut microbiota. For example, a recent study showed that the relative abundance of Firmicutes, Proteobacteria, and Bacteroidetes in the gut microbiota of Nlrp3KO and WT mice was substantially different38. Notably, fecal microbiome transplantation (FMT) from the Nlrp3KO mice alleviated stress-induced depressive-like behaviors in recipient mice. Chrna7KO mice, which have a deficiency in α7 subtype of nicotinic acetylcholine receptors (α7 nAChR), exhibited depression-like behavior and gut microbial changes . In a following study, FMT proved successful to transmit the depressive behavior to recipient mice via vagal nerve signaling40.MDD is a polygenic disease, and a most recent GWAS covering over 1.2 million participants identified 178 genetic risk loci and 223 independently significant single-nucleotide polymorphisms (SNPs) that were associated with the disorder at genome-wide significance51. Ample data point to poor-diversity and dysbiosis with respect to the abundance of Faecalibacterium and Bacteroides as possible traits and state-dependent characteristics of BD. Reduced butyrate production and richness also drives inflammation, which may be a previously unappreciated component of the pathophysiology driving BD52. Association also exists between gut microbial dysbiosis and the hypothalamic-pituitary-adrenal (HPA) axis dysregulation in BD, as a significant negative correlation between the count of Bifidobacterium and cortisol levels was recently found53. Notably, HPA axis dysfunction in BD may worsen intestinal permeability and gut microbial dysbiosis in turn. Supporting the role of gut dysbiosis in schizophrenia patients, colonization of Streptococcus vestibularis, a bacterium enriched in treatment naïve schizophrenia patients, causes social behavior abnormalities, and alters neurotransmitter levels in peripheral tissues in antibiotics-treated recipient mice54.GWAS reveals a genetic connection between schizophrenia and BD, and many of the risk genes have been linked to immunological response and inflammationFmr1 deficiency is likely to be caused by alterations in gut microbiota. More recently, gut microbiome changes involving Akkermansia, Allobaculum, Bifidobacterium, Odoribacter, Flexispira, Bacteroides, and Oscillospira have been profiled in Fmr1KO mice, and, of note, FMT from normal mice proved effective to mitigate autistic-like behaviors56.FXS is an inheritable neurodevelopmental disease characterized by autistic traits, such as mental retardation and impaired social communication or interaction. Recently, there is accumulating evidence that FXS caused by 57. ALS is thought to have a substantial genetic component with a high heritability, and many of the gene variations  that cause or predispose an individual to ALS have been identified58. In 2017, gut dysbiosis with a reduced population of butyrate-producing bacteria was reported in Sod1 (G93A) mice, which was rescued by butyrate treatment59. Indeed, novel evidence indicating a disease-modifying role for the gut microbiome has recently emerged, with certain gut microbial strains being causally linked to the behavioral symptoms in Sod1 and C9orf72 model mice61. Therefore, it is reasonable to dissect neuropsychiatric disorders from the perspective of reciprocal host gene-microbiome interactions.ALS is a fatal neurodegenerative disease that causes progressive motor neuron loss. ALS patients often show behavioral symptoms such as depression and anxiety20, unbiased identification of causal microbes in shaping neuropsychological diseases due to genetic variation is undoubtedly challenging. Here, we describe ongoing efforts and strategies seeking to answer this basic open question in microbiome study  genetic variant64. When healthy control and patients are carefully selected, such a microbe-phenotype triangulation strategy can be more generally applicable to human microbiome studies. However, this strategy has several caveats that may compromise the scalability. As a major concern, co-housing does not always result in intermediate phenotypes, and, instead, mono-directional transmission of one dominant phenotype could be observed65. Also, for rare species that may mediate a host phenotype, causal bacteria may be masked due to their relative low abundance66.Early pioneering studies by Gordon 63. For studies where co-housing is impractical, an alternative strategy would be dirty cage sharing, as recently shown by Guo et al.67. Specifically, in the search for key protective microbe in elite-survivors which were old male mice, a traditional cohousing approach with young male recipients might lead to fighting and injury to the older mice. By analyzing bacterial 16S rRNA genes in feces from donors and recipients after sharing dirty cages, it was shown that dirty cage sharing was effective in exchanging gut microbiota from donors to recipients.In essence, the microbe-phenotype triangulation strategy relies on the spatial transmission of causal microbes to enable comprehensive microbial analyses from microbially related miceet al. showed that both depletion of the gut microbiota by antibiotic cocktail and oral treatment with ampicillin alone limited the development of experimental allergy encephalomyelitis (EAE)68. In consistence, ampicillin-treated mice showed a unique microbiota structure in the small intestine, and a novel strain named operational taxonomic unit (OTU)0002 was the sole sequence that was almost completely depleted only from the small intestine of ampicillin-treated mice. Notably, mono-colonization with OTU0002 resulted in an increased severity of EAE symptoms and frequency of T helper 17 (Th17) cells, both in the small-intestinal lamina propria and spleen. In another study seeking to identify gut bacterial species that affect social activity69, mice were treated with different combinations of antibiotics, which showed that a microorganism exclusively sensitive to neomycin appeared to be responsible for modulating social activity and corticosterone levels after social stress. Further study identified Enterococcus faecalis  that promoted social activity and reduced activation of the HPA axis.Antibiotic cocktail comprising ampicillin, vancomycin, neomycin, and metronidazole is commonly used for assessing the role of gut microbiota in the host phenotype, although each antibiotic modulated the microbiota composition in a distinct way. Recent studies have also shown that the use of single antibiotics could also provide useful clues for identifying causal microbes. As an illustrative example, Miyauchi et al., for example, several important time points were selected for bacterial sequencing along with the disease progression, and then the results were further integrated with spatial transmission features60. Eleven distinct commensal bacteria that would increase or decrease within the disease process were subsequently identified, and the role of each strain was verified by single bacterial colonization into antibiotic pre-treated model group. This strategy is of special value for causal microbe identification from human microbiome samples, which is complicated with high interindividual variance70. For example, in the search for irritable bowel syndrome (IBS) subtype-specific microbial composition and metabolites, longitudinal sampling effectively limited heterogeneity seen in cross-sectional microbiome studies71. The results showed that stool microbiota composition exhibited greater variability over time in patients with constipation-type IBS (IBS-C) compared to diarrhea-type (IBS-D) cohorts. In another clinical study involving hematopoietic cell transplantation patients, daily changes in circulating neutrophil, lymphocyte, and monocyte counts and more than 10,000 longitudinal microbiota samples were profiled to reveal consistent associations between gut bacteria and immune cell dynamics overtime72. These strategies are readily applicable to screen causal microbes underlying behavioral disorders.Gut microbial structure shows inherent plasticity and dynamics. To strengthen the confidence of causal microbe identification, temporal patterns of the gut microbiota could be mapped for integration with spatial profiling. In the study by Blacher 73. This method is expected to enable reliable meta-analysis of metagenomic dataset from neuropsychiatry disease cohorts. Also, of note is the recent report of a machine learning-based framework that could jointly analyze paired host transcriptomic and gut microbiome data for deciphering host gene and gut microbiome interactions in disease74. This computational method can be applied to neuropsychiatric diseases for the identification of host gene-microbiome associations and key microbes that may influence behavioral outcomes.Computational algorithm is also finding a wide application on identifying causal microbes or biomarkers from clinical large-scale databases of microbial community. Batch inconsistence of multi-population metagenomic data presents a challenge in identifying robust microbial markers. Addressing this issue, recently an algorithm called Network Module Structure Shift (NetMoss) has been devised, which proved useful to identify shared and unique microbial biomarkers from a network perspective75.It should be noted that current microbial mapping techniques have limitations in providing high-resolution and functional information, especially given the difficulties associated with assigning gene-based labels and gene-function correlation. The commonly used 16S rRNA sequencing technique reveals the bacterial genes they carry and the compounds they produce or consume. Thus, a better approach might be identifying not just the taxonomy, but the metagenomic signature, a matrix describing which genes are stored in which bacteria, and what is their abundance. This fact suggests that the screening and validation of potential causal microbes should consider strain and host origin differences and the sequencing depth. Moreover, even in the same model, intestinal site, and mucosal region-dependent changes of gut microbial composition should receive due attention in sampling and data interpretation76. Concerning the neural transmission pathway, the message could be transmitted via the vagal or the sympathetic nervous system78. The effects of L. reuteri on social behavior were no longer present in vagotomized animals in a Shank3BKO mouse model of autism35. Also, via direct synapsing with vagal nodose neurons, enteroendocrine cells (EEC) have emerged as a new sensory mechanism for rapid transmission of gut signals79. More recently, in zebrafish, microbial, pharmacological, or optogenetic activation of Trpa1+ EECs proved successful to directly stimulate vagal sensory ganglia and activate cholinergic enteric neurons by secreting the neurotransmitter 5-hydroxytryptamine (5-HT)80. Microbial signals can also transmit through the sympathetic nerve. A gut-brain-gut neural circuit, which regulates efferent sympathetic tone and gastrointestinal transit has been recently identified78. This effect was mainly attributed to microbial metabolites, such as short-chain fatty acids and bile acids, as well as microorganism-modulated GLP-1 release.Current research in neurogenetics mostly seeks to answer how genetic variants regulate neuronal activity and circuit function in key brain regions using sophisticated cell-specific imaging, electrophysiology, and genetic manipulation techniques. Understanding how gut microbes communicate with the brain therefore holds another key to therapeutic target discovery. The past decade has witnessed huge advances in decoding the signaling mechanisms along the microbiota-gut-brain axis, which provide snapshots into the network connecting microbiome, brain and behavior including anxiety, cognition, nociception, and social interaction75, which appear to affect the brain in a direct or indirect manner. Elevated level of a microbial metabolite 4-ethylphenyl sulfate was previously reported in mice with social impairment47. A follow-up study showed that this gut-derived metabolite can enter the brain to affect oligodendrocyte function and neuronal myelin sheath pattern and promotes anxiety-like behavior in mice81. More recently, it is also reported that direct sensing of bacterial cell wall components (peptidoglycan) by hypothalamic Nod2+ neurons regulate appetite and body temperature in mice82. However, for those messengers that could not directly enter the brain, much more work is needed to fully address the exact mechanisms by which gut microbiota could distally impact on the brain and behavior. One common route is the sensing and transmission by neural pathway. For example, Lactobacillus brevis Bb14 (ATCC14869)-derived trehalose from sugar metabolism by the bacterial enzyme xylose isomerase activates peripheral octopaminergic neurons to regulate locomotor behavior in the fruit fly Drosophila melanogaster, while germ-free status or antibiotic treatment results in hyperactive locomotor behavior83. Recently, an inter-organ neural circuit for appetite suppression has been mapped, which conveys gut local GLP-1 signal to induce satiety84. Since gut microbes are intimately involved in the regulation of GLP-1 release, and GLP-1 has behavioral effects more than appetite control85, it would be of interest to further explore the involvement of this pathway in gut microbial regulation of behavior.Bidirectional gut-brain signaling can also occur via circulating hormones, metabolites, cytokines, and other neuromodulatory moleculessegmented filamentous bacteria (SFB), Bifidobacterium adolescentis, and Odoribacter splanchnicus are well known to induce IL-17 production. Notably, SFB-induced susceptibility to stress-induced depressive-like behaviors in mice were causally linked to the expansion of Th17 cells, which accumulated in the hippocampus86. Context-dependent (during embryonic brain development or in the adult brain) effects of IL-17 in social behavior has been previously reported87–89, and, more recently, mucosa-associated fungi was shown to promote social behavior in mice via sensory neuron IL-17 receptor86.The involvement of immunologic signal pathways in microbiota-brain interaction has also been reported. For example, gut microbiome members such as et al., which transplanted gut microbiota from human ASD donors into GF mice for mechanistic interrogation50. Through the inter-correlative analysis of species, metabolites, and behaviors of mice harboring human microbiota, candidate microbes, and metabolite that may mediate ASD behaviors were picked out for further validation. This strategy is also observed in profiling microbiota-host interaction in a Chd8± mouse model with ASD-like behavior, in which increased expression of amino acid transporters in the intestines of ASD mouse and high level of serum glutamine offer clues to understand the increased excitation/inhibition ratio in the brain34.Generally, the integration of ‘omics’ datasets, such as transcriptomics, metagenomics, proteomics, and metabolomics can be useful to pinpoint the regulatory microbes and signaling molecules underpinning host behavioral phenotype. This strategy is nicely depicted by the study of Sharon et al. to identify causal microbial metabolites in ALS from nicotinamide (NAM) and phenol sulfate, which had the highest metagenomic probability. By showing that phenol sulfate administration to mice did not relieve the symptoms of ALS mice, they focused on NAM for the following reasons: the marked differences in the metagenomic NAM biosynthetic pathway; the enrichment of NAM biosynthetic intermediates in serum upon supplementation with Akkermansia muciniphila (ATCC BAA-835); reduced abundance of genes from the gut-microbiome-derived tryptophan metabolizing pathway, which may be involved in NAM production; and the alteration of metabolites in the tryptophan pathway upon treatment with antibiotics or upon Akkermansia muciniphila supplementation60. Functional validation with NAM supplementation finally identified NAM as a key protective metabolite in ALS.Given the inherent noise in the omics data, ‘trial and error’ screening is necessary, and several more criteria are worthy of consideration. This is elegantly demonstrated by the study of Blacher 91, there is still limited insights into how the composition and function of gut microbial community are altered. In essence, this reflects the interaction between the host’s mutated genes and microbial genes that shape their fitness and survival in the gut, as shown by recent studies in mice. A balanced gut microbial configuration is characterized by the dominance of obligate anaerobic members of the phyla Firmicutes and Bacteroidetes, which prevents dysbiotic expansion of facultative anaerobic microbes, such as Enterobacteriaceae in part by limiting the generation of host-derived nitrate and oxygen. Epithelial mitochondrial bioenergetics and metabolism has therefore proven to be a canonical pathway by which host genetic signals remodel the gut microbial configuration. A typical sensor/mediator is epithelial peroxisome proliferator-activated receptor γ (PPAR-γ), which drives the energy metabolism of colonic epithelial cells toward β-oxidation and limits both the luminal bioavailability of oxygen and nitrate to prevent a dysbiotic expansion of potentially pathogenic Escherichia and Salmonella92. Mice lacking epithelial PPAR-γ signaling exhibited significantly elevated nitrate availability in the lumen and dysbiotic expansion of Escherichia coli that carries the genetic machinery for nitrate respiration93. Moreover, treatment with 5-amino salicylic acid, a PPAR-γ agonist, restored mitochondrial bioenergetics in the colonic epithelium, which alleviated dysbiosis triggered by high-fat diet and antibiotics92. A recent study has also established that intestinal hypoxia-inducible factor-2α (Hif2α) deficiency altered the balance of intestinal Bacteroides vulgatus to Ruminococcus torques to affect bile acid signaling and obesity-related insulin resistance94. Mechanistically, ablation of intestinal Hif2α reduced the level of epithelial-derived lactate, which enriched the polysaccharide utilization genes to promote the growth of Bacteroides vulgatus.Although host genetic variation is known to shape the gut microbial ecology95. Typically, secretory IgA directed against bacterial antigens has been proposed to shape intestinal microbial composition by multiple mechanisms, including inhibition of bacterial motility and reduction in bacterial fitness. As has been recently reported, activation of mechanistic target of rapamycin complex 1 (mTORC1) signal in CD11c cells alters IgA secretion at the mucosal site96. Reduced IgA production results in decreased gastrointestinal colonization of Lactobacillus johnsonii Q1–7, which in turn leads to lower food intake and body mass. In addition, another investigation has shown that mice with T cell-specific ablation of Myd88, an innate adaptor molecule, have defective development of T follicular helper cells in the gut, contributing to inappropriate IgA targeting of Clostridia and altered microbial balance97. This results in imbalanced expansion of Desulfovibrio at the expense of the loss of Clostridia, which contributes to increased lipid absorption and obesity susceptibility.Host genetics can also shape the commensal microorganisms by regulating the secretion of defense signals, such as antimicrobial peptides and immunoglobulin A (IgA)98. A recent study shows that loss of forkhead box O 1(Foxo1) in intestinal epithelial cells results in defects in goblet cell autophagy and mucus secretion, which induces gut dysbiosis, disruption of gut barrier integrity, and increased susceptibility to intestinal inflammation99. Deficiency G protein-coupled receptor 35 (Gpr35), previously known to regulate the activity of Na/K-ATPase and mitochondrial oxidative phosphorylation (OXPHOS) of epithelial cells100, was recently shown to induce goblet cell depletion and dysbiosis102. Interestingly, ablation of Calcitonin gene-related peptide (Cgrp) in nociceptor neurons or epithelial Ramp1 is also shown to reduce goblet cell mucus secretion, which induces dysbiosis and susceptibility to colitis103.Goblet cell secretion of mucin represents another way by which host genetics influence the symbiotic microbes104. Conclusive evidence that a genomic mutation is responsible for a specific behavioral phenotype can be obtained when littermate controls are used21. If wild-type control mice were provided from other animal facility, the influence of environment factors on gut flora was difficult to exclude. Of note, the fact that mice from different suppliers showed markedly variance in microbial background and biologic responses provides excellent clues for identifying key causal microbe, as demonstrated previously105.The interwoven gene-microbiota connection in neurobehavioral changes, as well as the limitations in current techniques, suggests some practical issues deserving attention in the effort to better understand the etiology and treatment strategy for neuropsychiatric disease. Firstly, given the strong impact of environmental factors on microbial composition, diet composition, and housing condition variance should be carefully considered in screening differential bacteria35. It is reasonable that host impacts of gut microbial change could be attributed to the interactions among different microbes. Indeed, in the study of Miyauchi et al., mice mono-colonized with OTU0002 exhibited EAE symptoms that were less severe than those of specific-pathogen-free (SPF) mice, which indicated that other bacterial members of the microbiota could also be participating in the pathogenesis of EAE. In the search for potential microbial interactions, they further showed that two distinct signals from OTU0002 and a L. reuteri strain  coordinately activate autoreactive T cells in the small intestine to drive autoimmune responses. In practice, to better mimic microbial interaction network in host regulation, a simplified microbial community consisting of representative high-abundance bacterial species would provide more physiologically similar, while structurally defined microbial models for the validation of key causal microbes. This strategy is readily found in the study of Lobel et al.106 and Kasahara et al.107, which support that the colonization of the structurally defined consortium of bacteria could provide physiologically relevant and therefore more strong insights into microbe-host interactions.Secondly, in mono-colonization studies, it would be necessary to profile whether the global microbial community would be affected to understand whether the host impact may derive from microbe interactions. Indeed, studies have shown that colonization of a single strain may elicit microbiome-wide changesDrosophila melanogaster, and Caenorhabditis elegans are useful for gut-brain-microbiota studies related to neuropsychiatric diseases108. The accumulated expertise in genetic and microbial manipulation and human-relevant neurobehavioral parameters could become advantages for gaining holistic insights into gene-microbiota-behavior interplay and the signaling basis. Some elegant studies are available in relation to the dissection of microbial connection to sensory and locomotive behavior109.Thirdly, although we largely focus on data from mice models, non-mouse model organisms, such as zebrafish, 110. In particular, through Mendelian randomization and mediation analyses, this study uncovered putative causal relationships between the gut microbiome and plasma metabolites. Application to neuropsychiatric disorders would be expected to clarify robust microbial features to clinical diagnostic and preventive purposes.Lastly, in the clinical setting, age, sex, dietary habits, co-morbid diseases, and drug exposure are common confounding factors for identifying causal links. Addressing these inter-individual variability factors is crucial to elucidating the genetic and microbial factors that causally shape human neuropsychiatry diseases. A recent prospective large-population study dissecting the influence of the gut microbiome, diet, and genetics on plasma metabolome variation presents a useful workflow to tackle this issueCurrent therapeutic approaches for neuropsychiatric disorders aim to target the brain directly. The accumulating findings on host genetic-gut microbial interplay in neuropsychiatric diseases broaden our views on the factors shaping individual’s susceptibility to mental illnesses and underscore the bidirectional gut-brain crosstalk in behavioral control. An emerging picture is that microbial and host genetic factors may control different aspects of brain diseases, which provide the rationale for combinatorial targeting of genetic factors and microbial signaling for comprehensive intervention of complex behavioral diseases. Deciphering gene-microbiome-phenotype trialogue in complex neuropsychiatric diseases may therefore inspire novel therapeutic strategies and precision intervention. However, since most of the causal data are from animal studies, the results still should be approximated to humans with caution.112. Neural circuitry allowing gut microbial control over behaviors remains to be fully unmapped, under both physiological and pathological conditions. Advances in this field increasingly benefit from the technical advances in optogenetics, chemogenetics, and virally delivered molecular tools for neural manipulation in an organ-specific and inter-organ manner113. Progress in this field could be integrated with genetic manipulation studies to offer combinatory targets for future drug discovery. Specifically, the microbial signals or microbe-responsive pathways promoting inner homeostasis or resilience to stressful events could be strengthened as a preventive or therapeutic strategy.The field of microbiome research is experiencing a progression from associative linking to causal and mechanistic insights, which presents new opportunities to decipher how this dynamic community is functionally implicated in the genetic predisposition to neuropsychiatric diseases in concert with other environmental factors such as diet and lifestyle. In future studies, the causal relationship between genetic variance, key microorganisms, and behavioral changes remains to be fully elucidated in the clinical setting in order to provide novel diagnostic biomarkers and therapeutic targets. Another key question is how the microbes achieve central relays of local signals to specify the behavioral changes. Research insights into the communication pathways between the gut and the brain is critical to drive this frontier forward. Meanwhile, elucidation of the key biologic synthetic pathways of microbes and the sensors for the signaling molecules could provide potential drug targets. Recently, the devise of novel techniques for genetic manipulation of gut microbes and targeted protein degradation in bacteria could enable efficient clues to answer these key questions


Rheumatoid arthritis (RA) is a complex condition that displays heterogeneity in disease severity and response to standard treatments between patients. Failure rates for conventional, target synthetic, and biologic disease-modifying rheumatic drugs (DMARDs) are significant. Although there are models for predicting patient response, they have limited accuracy, require replication/validation, or for samples to be obtained through a synovial biopsy. Thus, currently, there are no prediction methods approved for routine clinical use. Previous research has shown that genetics and environmental factors alone cannot explain the differences in response between patients. Recent studies have demonstrated that deoxyribonucleic acid (DNA) methylation plays an important role in the pathogenesis and disease progression of RA. Importantly, specific DNA methylation profiles associated with response to conventional, target synthetic, and biologic DMARDs have been found in the blood of RA patients and could potentially function as predictive biomarkers. This review will summarize and evaluate the evidence for DNA methylation signatures in treatment response mainly in blood but also learn from the progress made in the diseased tissue in cancer in comparison to RA and autoimmune diseases. We will discuss the benefits and challenges of using DNA methylation signatures as predictive markers and the potential for future progress in this area. Rheumatoid arthritis (RA) affects around 0.5–1% of the global population. It is characterised by synovial joint inflammation that leads to pain, swelling and reduced mobility and, if poorly responsive to therapy, irreversible damage to cartilage and bone. The aetiology of RA is unknown, but good progress has been made in elucidating the pathogenetic mechanisms. Monozygotic twin studies show a concordance rate of ~12–15% . Over thIn the pathogenesis of RA, the combination of environmental triggers and underlying genetic susceptibility causes the activation of an autoimmune response in the synovium which becomes inflamed, resulting in synovitis. This inflammation is characterized by the activation of immune cells including T-cells, B-cells, macrophages, and the release of pro-inflammatory cytokines, including tumour necrosis factor-alpha (TNF-α), Interleukin-1 (IL-1), Interleukin-6 (IL-6) and Interleukin-17 (IL-17). These cytokines play a pivotal role in perpetuating the inflammatory response and promoting joint damage . The resAlthough standard therapeutic regimens vary between countries, current first-line therapy consists of conventional synthetic DMARDs (cs-DMARDs), i.e., methotrexate (MTX), sulfasalazine, hydroxychloroquine and leflunomide, as monotherapy or in combination and with corticosteroids if clinically indicated . The mosSecond-line therapy consists of biologic DMARDs (bDMARDs), which are monoclonal antibodies to specific key cytokines or cytokine receptors including TNF-α and IL-6, or cellular targets such as CD20 and CD80/86 . ClinicaClinical parameters including lower disease activity at baseline measured by the Disease Activity Score of 28 joints (DAS28) and not smoking were found to be weakly predictive of treatment response with MTX . Male sep ≤ 5 × 10−5 [p = 10−8) for any outcome measure [p = 10−7) and this study did not replicate any findings from Senapati et al. Few genetic studies have been conducted specifically for analysing response to sulfasalazine, hydroxychloroquine, or leflunomide. For leflunomide IL-6 -174G/C polymorphism was found to be associated with response. The GG genotype confers a higher risk of therapeutic failure than GC or CC [Initial research into markers of MTX response focused on genetic studies with mixed results. Senapati et al., conducted the first genome-wide analysis of MTX treatment response in RA patients. From a cohort of 457 patients, they found 10 new risk loci for poor response using a significant cut-off 5 × 10−5 . In a la measure . The strGC or CC . More recently, transcriptomic signatures associated with prognosis and treatment response to csDMARD have been reported. For example, Humby et al., demonstrated that specific gene expression patterns in the synovial tissue at baseline in treatment naïve RA patients can predict the extent of radiographic joint damage after 12 months following csDMARD treatment. Ongoing joint damage is considered an indicator of treatment non-response. The prediction model incorporates rheumatoid factor titre, and the expression level of seven genes using NanoString probes applied to synovial biopsies: SDC1 (encodes plasma cell marker CD138), CSF2 (stimulates growth and differentiation of multiple immune cell lines including granulocytes and macrophages), DENND1C , CD180 (mediates innate immune response and activates NF-kappa-B), UBASH3A (induces apoptosis in T-cells), CXCL1 (chemoattractant for neutrophils), MMP10 . The predictive algorithm had an area under the curve (AUC) of 0.88 , which sIn a separate analysis of the same cohort of treatment naïve RA patients, Lewis et al., used RNA-Seq to show that expression levels of specific groups of associated genes (gene modules) in the synovial tissue were associated with response to csDMARDs . SpecifiHowever, csDMARD treatment in the above studies varied significantly. Patients were treated according to the British Society of Rheumatology’s guidelines and received a diverse range of csDMARD treatments/combinations according to physician and patient preferences. Although these results are promising we are still far from developing a clinically useful test for predicting individual response to specific csDMARDs. Research into biomarkers of bDMARD response also initially focused on genetics. Multiple loci and SNPs associated with response to TNFi in RA patients have been identified in recent years, but these findings have not been consistently replicated in different populations. For example, in the PTPRC gene encoding receptor tyrosine-protein phosphatase C or CD45, the rs10919563 G>A polymorphism has been associated with reduced efficacy of the TNFi: adalimumab, etanercept, and infliximab . HoweverTranscriptomic signatures associated with bDMARD response have been found ,44. In aMultiple gene sets have been identified in whole blood, which are associated responses to TNFi including infliximab ,46 and aJak inhibitors (JAKI) are a class of target synthetic DMARD that were first introduced for widespread clinical use by the approval of tofacitinib by the FDA in 2012. Research into JAKI has mainly focused on their efficacy, mechanisms of action and safety profile. Investigations into biomarkers of response to Jak inhibitors lag the other DMARDs due to the relatively shorter period they have been in use. Valli et al., found that tofacitinib treatment reduced both the DAS28 score and the levels of certain circulating pro-inflammatory markers, the most pronounced reduction being in IL-6, C-X-C motif chemokine ligand 1 and matrix metalloproteinase-1. Additionally, higher baseline circulating levels of IL-6 and lower levels of C-C motif chemokine 11 were associated with DAS reduction post-treatment . HoweverThese results show that there are distinct synovial transcriptomic profiles of both response and non-response to csDMARDs and specific bDMARDs. However, genetic and transcriptomic studies only provide insight into one aspect of the biological mechanisms underlying response. Genetic variation affects epigenetic modifications including DNA methylation, which in turn affects gene transcription. Therefore, DNA methylation studies should help to further elucidate the biological mechanisms of response. Many recent studies have focused on DNA methylation as a biomarker of RA treatment response with a strong, growing body of evidence that DNA methylation plays an important role in both pathogenesis of RA and treatment response.Epigenomics refers to modulations that influence gene expression but do not change the underlying genetic code. These include DNA methylation, histone modification, and microRNA modulation . This reAs schematically illustrated in CpG sites exist across the entire genome in both coding and non-coding regions but CpG sites then cluster in groups called islands. A total of 70% of islands are found in promoter regions of genes , and 50%DNA methylation patterns are highly tissue and cell-type-specific, which reflects its essential role in normal development. DNA methylation enables selective temporal activation of lineage-specific genes, and suppression of pluripotency genes in the early embryonic stages, which ensures proper establishment of gene expression patterns for specific tissue and cell type development . DNA metThere has been significant progress in recent years in DNA methylation research in RA showing that the methylome of patients displays distinct differences compared with healthy controls. Liu et al., performed the largest epigenome-wide association study to date on the whole blood samples of 354 ACPA-positive RA cases and 337 controls . Their rRecent studies into the methylation pattern of specific immune cell types in RA including fibroblast-like synoviocytes (FLS), monocytes and lymphocytes have also revealed interesting insights into RA pathogenesis. For example, peripheral blood mononuclear cells (PBMCs) of RA patients were found to be globally hypomethylated compared to healthy controls , while aJUN, STAT1, PTEN, and CD44 genes exhibit hypermethylation, while KRAS and ALB show hypomethylation. Gene ontology (GO) enrichment studies indicate that the differentially methylated genes in RA are connected to T-cell biological processes, suggesting that DNA methylation plays a role in regulating CD4+ T-cell function in RA [UBASH3A, a gene involved in antigen presentation to T-cells was found [CTLA-4 promoter region leading to reduced production of CTLA-4, which was associated with compromised Treg activity in RA [DNA methylation patterns in the cluster of differentiation 4 positive (CD4+) T-cells of RA patients are different compared to healthy controls. In RA patients, on in RA . Comparias found . T-cellsas found ,72. Aberas found . Cribbs ty in RA . CASP1—encodes Caspase 1 that induces cell apoptosis, MAP3K5 a MAP kinase involved in the innate immune system, STAT3 a transcription activator, activated in response to cytokine signalling, MEFV (pyrin) that generates inflammation in response to interferon-gamma signalling. Hypomethylated CpG sites were primarily located on gene pathways involved in cell adhesion/migration and extracellular matrix interactions [PTEN gene promoter region has also been shown to activate FLS in RA pathogenesis [Studies of FLS in RA patients showed significant differential methylation compared with osteoarthritis (OA) patients . Nakano ractions , also suractions ,78. Interactions , suggestractions . DNA metogenesis . These sAnalysis of immune cells from the blood and synovium of RA and OA patients showed the largest differences in methylation were between different tissues, rather than between disease states . This suSingle-cell RNA sequencing analysis of cells derived from synovial tissue has found distinct subpopulations of inflammatory cells and fibroblasts that are not present in the blood . Within Histological studies of immune cells in the synovial tissue of treatment naïve early RA patients found three distinct pathotypes: a lympho-myeloid type dominated by the presence of B-cells and myeloid cells, a diffuse-myeloid type myeloid cells but very few B-cells and the pauci-immune type characterised by scanty immune cells and prevalent stromal cells . The lymDNA methylation analysis of synovial tissue is still in its infancy but, as the methylation process is highly cell-specific, the methylation status of synovial tissue will naturally reflect its diverse cellular composition as per the above-described pathotypes. Consequently, each pathotype is likely to have a distinct DNA methylation profile and it would be interesting to establish whether specific synovial DNA methylation signatures will enhance our future ability to predict prognosis and treatment response in RA.Research into DNA methylation in other autoimmune inflammatory diseases is still in the early stages but there have been some promising results. A systematic review of methylation in inflammatory bowel disease found consistent differential methylation was identified for 256 DMPs in the peripheral blood of IBD patients compared to healthy controls . DNA metDNA methylation has great potential as a biomarker because it is dynamic and constantly modified in response to stimuli. It is more stable than gene expression at transcript and protein levels and is inherited between cell divisions . The mosHowever, using DNA methylation as a biomarker also poses certain challenges. Age, sex, smoking, alcohol consumption, diet, stress, and exposure to environmental chemicals can all induce changes in DNA methylation patterns . These cAs previously discussed, genetic and transcriptomic signatures linked with treatment response in RA have been found. As gene expression is regulated by DNA methylation, it is possible that DNA methylation signatures linked to csDMARD response also exist and might provide a more accurate biomarker as DNA methylation is more stable than gene expression levels. So far, all DNA methylation studies exploring associations/predictability of response have been performed on whole blood or cells isolated from blood, as this is the most accessible form of tissue for investigation. Most studies investigating the relationship between DNA methylation and csDMARD response centre on MTX as a monotherapy or in combination with steroids and other csDMARDs . Recent In brief, MTX treatment has been shown to change global methylation patterns in important immune cells. De Andres et al., found treatment naïve RA patients have global hypomethylation of blood T-cells and monocytes compared to healthy controls . QuantitGlobal methylation patterns have been shown to be correlated with clinical response. Gosselt et al., investigated the global methylation of leukocytes from 181 RA patients treated with MTX or MTX and 2 other csDMARD. They discovered that higher baseline global DNA methylation was associated with smaller decreases in DAS28 CRP from baseline and with MTX non-response after 3 months of treatment . LieboldADAMTSL2 (encodes a secreted glycoprotein that interacts with the extracellular matrix) and BTN3A2  were very strongly associated with treatment response. Increased methylation at these sites in combination was the biggest predictor of response  [p value of 1 × 10−6 instead of standard FDR correction. Four additional DMPs at the 4-week time point predicted an improvement of swollen joint count and CRP at 6 months. Gosselt et al., found no DMPs or differentially methylated regions (DMRs) at genome-wide significance level in pre-treatment PBMCs between 68 responders and non-responders to MTX monotherapy or combination therapy [DNA methylation at specific CpG sites has been linked to csDMARD response. Glossop et al., extracted peripheral blood samples from 46 cs-DMARD naïve RA patients, who were subsequently treated with MTX, hydroxychloroquine, or sulfasalazine. Genome-wide DNA methylation profiling of T-cells found six statistically significant DMPs between responders and non-responders after FDR correction. Two specific CpG sites located on the genes: ificity) . Nair etificity) . However therapy . The disAlthough the results from these studies are interesting, there is no consensus on DNA methylation patterns of csDMARD response and more research needs to be conducted to find a reliable methylation biomarker of response. Additionally, most studies look at a combination of different csDMARDs so it can be difficult to understand the DNA methylation patterns associated with response to specific agents. Monotherapy studies primarily focus on MTX and to date, there are no specific DNA methylation studies of response to monotherapy with sulfasalazine, leflunomide, or hydroxychloroquine, most likely because there are relatively few patients on monotherapy with these agents. DNA methylation analysis of synovial tissue is a more promising future avenue of investigation compared to blood. Transcriptomic signatures of response have been found in the synovial tissue , and theResearch into DNA methylation patterns associated with biologic response is still at a relatively early stage, with only a handful of studies published confined to the peripheral blood . To dateLRPAP1 gene on chromosome 4. This gene encodes a protein that interacts with the low-density lipoprotein (LDL) receptor-related protein and facilitates its proper folding and localization. It is not known to be involved in immune response or inflammation and the exact role of methylation at this locus in RA is still unclear. Methylation quantitative trait loci analysis was carried out on the LRPAP1 loci. The A allele of rs3468 SNP correlated with higher methylation levels at the top two DMPs and increased risk of poor response. SNP rs3468 was analysed in an independent cohort of 1,204 TNFi-treated RA patients and each stepwise increase in the A allele from GG to AA caused a 1.28-fold increased risk of being in the poor response group. This suggests genotype affects DNA methylation at key positions, and the combination of these factors influences individual treatment response. However, a longitudinal analysis of RA patients treated with TNFi conducted by Julia et al., found no genome-wide significant DMPs at baseline between responders and non-responders [p value threshold to pass FDR also increases and therefore probes that were statistically significant from the 450K array may not be significant when tested with all the probes from the EPIC array. Specific DNA methylation signatures of response have been identified by Plant et al., in an epigenome-wide association study on pre-treatment whole blood samples from 72 etanercept-treated RA patients . Five Cpsponders . This stsponders . The dissponders  and in tp < 0.05) DMPs between response groups but none at the genome-wide significance level. Globally more hypermethylated DMPs were found in etanercept responders compared to adalimumab responders, suggesting there is also a distinct DNA methylation pattern of response for each drug. Using gene expression data from PBMCs, monocytes and T-cells and the DNA methylation data from PBMCs, the investigators built a predictive algorithm using machine learning. The model for predicting etanercept response using DNA methylation had an overall accuracy of 88%, which surpassed the accuracy of pure gene expression models (73% to 79%). The adalimumab DNA methylation model had a high overall accuracy of 84%, which was similar to the pure gene expression model using DEGs from PBMCs (85%). This suggests that DNA methylation patterns may be used to generate accurate predictive algorithms for TNFi response. Further research into integrating DNA methylation, transcriptomic and genotype data into one predictive model may produce more accurate algorithms. Tao et al., investigated gene expression and DNA methylation patterns associated with etanercept and adalimumab response . DiffereDNA methylation signatures have shown great promise as biomarkers for diagnosis and predicting treatment of cancer. In contrast, DNA methylation research in RA is still in the early stages and no reliable biomarkers of treatment response have been found. The major challenge facing this area of research is the relatively small methylation differences between treatment groups in RA. Methylation variations of <5% are the norm for non-cancerous tissue  and largFurther challenges facing methylation research in RA treatment response relate to the lack of samples from the diseased tissue, i.e., the synovium. All DNA methylation studies of treatment response to date have used peripheral blood samples but the blood methylome is affected by many different factors, including other causes of inflammation independent of RA. In contrast, the methylation patterns of the synovium and synovium-derived cells may more accurately reflect the biological mechanisms underlying RA-specific inflammation.DNA methylation analysis of the whole synovial tissue and/or single cells derived from synovial tissue digestion is the logical next step and is more likely to provide specific biomarkers for RA treatment response in the future. A study comparing gene expression (RNA-seq) in the synovium versus the blood of RA patients showed much greater differential gene expression in the synovium compared to the blood . In thatMethods for generating DNA methylation data have progressed significantly. The Illumina BeadChip arrays—450K and EPIC, are relatively new but increasingly used for epigenome-wide association studies . These aFinally, DNA methylation should not be viewed in isolation as it is only one type of epigenetic modification. Histone modifications and microRNAs also contribute to the pathogenesis of RA, in part through their regulation of DNA methylation . FurtherIn conclusion, the emerging evidence from numerous studies highlights the potential of DNA methylation as a promising biomarker for predicting the response to treatment in patients with RA. DNA methylation alterations have been observed in key genes and regulatory regions associated with immune system dysregulation and inflammatory pathways in RA. These epigenetic modifications, particularly in genes involved in B-cell/T-cell differentiation and cytokine signalling, appear to play a crucial role in pathogenesis and treatment response.Although substantial progress has been made, further research is required to validate the utility of DNA methylation as a reliable biomarker of treatment response in RA. Large-scale prospective studies, including diverse patient populations and different treatment regimens, are warranted to establish robust associations and refine predictive models. Additionally, investigating the dynamic nature of DNA methylation patterns throughout the course of treatment will provide valuable insights into the underlying mechanisms of therapeutic efficacy. DNA methylation holds significant promise as a biomarker for predicting treatment response in RA. Its potential to provide valuable insights into disease mechanisms and guide personalized therapeutic approaches makes it an exciting area of research in the field of rheumatology. Future advancements in our understanding of DNA methylation dynamics and its functional consequences will undoubtedly contribute to improved patient outcomes and the development of precision medicine strategies for RA.


Paenarthrobacter nicotinovorans was isolated and screened for antimicrobial activity. Many promising antibiotic candidates are not ultimately used because of toxicity. After screening secondary metabolites for antimicrobial activity, they were screened for cytotoxicityin vitro in the human HeLa cell line. The results show that there is no detectable cytotoxicity in HeLa cells.In a previous study, Paenarthrobacter nicotinovorans were displaying antimicrobial activityin vitro. The CytoTox-Fluor™ Cytotoxicity Assay was utilized to test the cytotoxicity of increasing amounts of the secondary metabolites as compared to the toxicity of digitonin, methanol, and water  were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1x Penicillin-Streptomycin-Neomycin (PSN).Toxicity testingPaenarthrobacter nicotinovoransbacteria to the HeLa cells was measured by using a commercial CytoTox-Fluor™ Cytotoxicity Assay  according to the manufacturer’s instructions. The positive control was 300ug/ml DigitoninPaenarthrobacter nicotinovorans(10mg/ml) was used at various concentrations in the toxicity experiments, and various incubation times were used to determine if concentration or incubation time increased toxicity. Ten thousand Hela Cells per well were used in a 96-well plate and the fluorescence measurements were obtained using the Varioskan™ LUX (ThermoFischer) multimode microplate reader with the rhodamine-110 (485nmEx/520Em) filter set.The toxicity of the secondary metabolites from theStatisticsMicrosoft Excel was used to analyze the data. Student T-tests were utilized to determine the level of significance . All experiments and samples were run in triplicate.


Despite the great success of vaccines over two centuries, the conventional strategy is based on attenuated/altered microorganisms. However, this is not effective for all microbes and often fails to elicit a protective immune response, and sometimes poses unexpected safety risks. The expanding nano toolbox may overcome some of the roadblocks in vaccine development given the plethora of unique nanoparticle (NP)-based platforms that can successfully induce specific immune responses leading to exciting and novel solutions. Nanovaccines necessitate a thorough understanding of the immunostimulatory effect of these nanotools. We present a comprehensive description of strategies in which nanotools have been used to elicit an immune response and provide a perspective on how nanotechnology can lead to future personalized nanovaccines. Nanoscale materials can extend antigen stability, enhance immunogenicity, and improve antigen presentation time in the targeted cell or tissue.The reasons behind the current success of advanced nanoscale vaccine technologies and how they differ from traditional and conventional vaccines in terms of immune modulation are discussed.The capacity and extent of eliciting humoral and cell-mediated immune responses by nanovaccines are reviewedWe present a list of all currently FDA-approved nanovaccines and those in clinical trials.An in-depth and rational understanding will be necessary for the development of nanotools for use in future vaccines. We overview the lessons learnt from this potentially transformative nanovaccine development and how they have been used to elicit an immune response, with a focus on the most recent nanovaccines. Box 1Extended antigen stability: because of the protective nature of the NPs, the antigens are protected from degradation by cellular components and enzymes.Enhanced immunogenicity: the NPs themselves can be immunogenic, leading to an enhanced immune response against the target antigen.Targeted delivery: nanovaccines can be designed to deliver antigen to targeted sites such as specific cell types or tissues, and thus reduce the likelihood of harmful side effects.Protection of antigens and adjuvants against enzymatic and proteolytic degradation: key immunogenic components such as peptides, oligonucleotides, and adjuvants are protected from degradation by the nanovaccine formulation.Evoke both humoral and cell-mediated immune responses: the two major branches of immunity (the antibody and cellular responses) can both be enhanced by nanovaccines.Present multiple components in a single platform: multiple antigens can be included in the same NP, leading to a nanovaccine formulation that can potentially protect against a wider range of antigens or infections.Enhanced duration of antigen presentation and DC processing: professional APCs require time to recognize and process antigen before presenting it to elicit a downstream immune response. Nanovaccines can persist for a longer time without alteration or degradation and thereby provide ample opportunity for APCs to boost the immune response.Alt-text: Box 1The immune system is an interconnected mesh of cells, tissues, and organs that protect the body against fatal diseases. Immune homeostasis is disrupted by either an underperforming or hyperactive immune response; the former can fail to protect against a simple infection  whereas NPs can provide strong protection to both the antigens and adjuvants against enzymatic and proteolytic degradation . NPs canNPs are ideal vehicles to deliver antigens for vaccination because they are comparable in size to viruses and have the ability to load and release active biomolecules . Many tyNanovaccines exploit NP drug delivery systems in general, and biocompatibility and safety are major metrics. Although the goal of nanovaccines is to elicit a specific immune response, it is important that their immunogenicity is antigen-specific rather than NP-specific . By contAntigens delivered by NPs are known to elicit stronger antigenic responses compared to their free counterparts because of the combination of enhanced stability, sustained release, and adjuvant effects , 59.. NPFor example, 1000 nm bovine serum albumin (BSA)-loaded PLGA particles evoked a more robust serum IgG response than particles sized 200–500 nm . By cont+ T cells and interferon (IFN)-γ production [in vivo animal model where polystyrene beads of 48 nm covalently bound to antigen induced an enhanced antigen-specific Th1-biased response and IFN-γ production [+ T cells [Another study showed that nanobeads of 40–49 nm could evoke the secretion of Th1-biased cytokines, whereas nanobeads of 93–101 nm elicited Th2-biased cytokine secretion following immunization in mice . These ooduction . This waoduction . Other s T cells ,82. Rese T cells . Given s T cells ,84,85. P+ T cell response [Antigens of interest can be either encapsulated within or attached to the surface of NPs. Antigen encapsulation can be achieved with polymeric, micellar, and liposomal NPs , and surresponse . When chresponse  must be in vitro internalization was achieved by the encapsulation architecture; however, the difference might be caused by a change in surface charge [To date, there are limited systematic studies on the effect of loading methods on nanovaccine efficiency. One reason is that the loading method is often specific to the NP system of choice, such as surface functional groups, geometry structure, and fabrication technique . It was e charge . In addie charge . Severale charge , 99.. Foe charge . In DNA e charge ,102). ToAntigens delivered by NPs are internalized through several endocytic pathways. Apart from the size effect discussed above, surface charge and surface functionalization of targeting molecules can facilitate delivery to APCs for antigen presentation. Cationic NPs are internalized by APCs more rapidly and usually promote intracellular trafficking through endosomal escape . Cationi+ and CD8+ T cell responses [+ and CD8+ T cell responses, and generated stronger CD8+ T cell response than the soluble antigen and LPS mixture [+ T helper cell activation pathway, DCs can also process antigens and present them via the MHC I pathway leading to activation of CD8+ T cell response in a process known as 'cross-presentation' [+ T cell expansion by antigen cross-presentation [+ T cell expansion, whereas non-degradable linkers do not [DCs express cell-surface mannose receptors which help in antigen internalization through mannosylation, and this enhances the activation of CD4esponses . The sam mixture . By targ mixture . In addintation' ,109. Thientation ,111. A sentation . Cross-ps do not .in vitro in comparison to the soluble antigen. Similarly, antigen displayed on the surface of multilamellar vesicles provided an enhanced humoral response compared to the encapsulated antigen. However, studies are few and further exploration is warranted. Elevated levels of antigen-specific antibodies can also be achieved by multivalent presentation of antigens, and NP systems can serve as the platform for this purpose. Ueda and colleagues have engineered self-assembling NPs for tailoring the optimal geometry for multivalent presentation of viral glycoproteins [Other than the cell-mediated immune response, various nanovaccines can elicit humoral responses. B cells, which oversee antibody production, require prolonged and constant activation to generate humoral responses. As mentioned previously, the strategy for loading the antigens onto the NPs may have a profound influence on the resulting humoral responses. For example, calcium phosphate NPs with the antigen covalently attached to the surface exhibit a substantial increase in B cell activation proteins .in situ concentration of LN-resident DCs is extremely high [The generation of a cell-mediated immune response relies on efficient trafficking or drainage of antigenic components to LNs for further processing and presentation to T and B cells. LNs thus represent a crucial target site for the delivery of vaccines and other immunotherapeutic agents because direct delivery of antigenic components into APCs residing in LNs can induce more potent and robust immune stimulation than can antigen uptake by migrating APCs. LNs also contain a substantial fraction of resident DCs which are phenotypically immature and well equipped for simultaneously internalizing antigens and particles . By targely high ,118. TheAs mentioned in the previous section, particle size plays an important role in LN targeting and retention. In one study, a synthetic vaccine NP (SVNP) was developed to improve the targeting and retention efficacy of cancer vaccines . The posNanomaterials are known to boost the immune system and have been used to develop vaccines when conjugated with antigens. We review here cases of inflammation reported in the literature that resulted from inflammatory cytokine release following NP administration. The Th1 or Th2 responses elicited thus caused either an efficient immune response or damage to the host tissue.Mycobacteriumtuberculosis (MtB) lipids attached to chitosan NPs induce both cell-mediated and humoral immunity leading to enhanced secretion of IgG, IgM, and Th1/Th2 cytokines [in vivo [The use of a lipid-based particle (ISCOMATRIX) as the adjuvant for a chimeric peptide vaccine containing multiple epitopes of T cell lymphotropic virus (HTLV) type I led to enhanced production of mucosal IgA and IgG2a antibody titers as well as increased IFN-γ and IL-10 production . Carbon ytokines . Amantad[in vivo .+ A20 nanovaccine maintains immune homeostasis by suppressing Th2 inflammation and promoting the regulatory T cell (Treg) response and IL-10 production in lung airway tissue of an allergic asthma murine model [Multiwalled carbon nanotubes and silica NPs can both activate the NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) inflammasome leading to uncontrolled pathological inflammation. Superparamagnetic iron oxide NPs (SPIONs) showed enhanced activation of inflammatory genes in response to LPS . The PLGne model .Synergistic stimulation of the production of IL-1β by some NPs and bacteria induces strong pathological inflammation leading to leukocyte influx, swelling, fever, vasodilation, and inflammation-driven tissue damage . ElevateSize-dependent immunogenicity of polystyrene particles carrying CpG oligonucleotides was observed in DCs leading to differential expression of IL-6 and IFN-α. CpG-mediated activation of the MAPK and nuclear factor κB (NF-κB) pathways induced the expression of proinflammatory cytokines  . The Th1+ T cell memory after vaccination [+ T cells [+ T cell more effectively following antigen delivery [Potential cytotoxicity of CTLs was observed in an overtly activated proinflammatory cytokine  response following albumin/albiCpG nanocomplex inoculation into mice. Encapsulated OVA polyanhydride NPs boosted the formation of antigen-specific CD8cination . Subcutacination . Hyperac T cells . More efdelivery .Only a few nanovaccines have been successfully translated from the laboratory to the clinics. Of these, most only elicit humoral responses, and there is an unmet need for the development of vaccines that can generate strong cellular responses against infectious diseases and cancer. Vaxfectin® is a cationic liposomal nanovaccine which is currently in clinical trials. Vaxfectin® has been used against herpes simplex virus type 2 (HSV-2) and also against influenza virus (H5N1) . SimilarSignificant attention to NPs has been recently drawn during the development of effective vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) . SynchroBoston-based Moderna in conjunction with the National Institute of Allergy and Infectious Diseases (NIAID) developed a mRNA-based NP vaccine against SARS-CoV-2 . The mRNIn addition to SARS-CoV-2, the use of nanovaccines is widespread in multiple other diseases as well. Many of them have been approved by FDA and/or European Medicines Agency (EMA) and others are currently in clinical trials. A list of such vaccines is provided in in vivo, toxicity, and the need for multiple booster immunizations. Nanovaccines, which are the focus of this review, provide distinct advantages over conventional vaccines because of their size proximity to pathogens, controllable physicochemical and biophysical attributes, enhanced protection of the antigen from degradation, biopersistence, improved transport through the lymphatics and into LNs, and codelivery of immunomodulatory molecules to boost immune recognition  during the development of different coronavirus disease 2019 (COVID-19) vaccines. Nanotools can offer a range of advanced strategies to potentially develop a seasonal vaccine where one infection may possibly facilitate other infections . It remains to be determined whether nanovaccines with multiple epitopes and/or adjuvants can be developed to generate broad-spectrum immunity. Nanotools offer the best possible non-viral strategy to encapsulate and deliver nucleic acids (including plasmids and mRNA), although thermostability remains an unresolved issue. It is well established that the immune system is differently configured in different individuals, and a 'one-size-fits-all' approach is not an optimal solution – nanovaccines hold potential in the development of a new frontier of personalized vaccines that will ensure wider and longer-term protection .Figure IAlt-text: Box 2Despite advances in the development of traditional vaccines, improvements are needed because of the weak immunogenicity of conventional vaccines, intrinsic instability ognition , Box 2.BRecent advances in nanoengineering have played a pivotal role in developing the highly anticipated liposome-based mRNA vaccine against SARS-CoV-2. Nevertheless, there are unanswered challenges in the path of successful translation of various nanovaccines. The nanoscale size range of the antigen vehicle is a crucial criterion which determines the spatial location of the antigen. The optimum size is not generalizable and depends on several factors such as the chemical composition of the nanovaccine and opsonization by complement and complement receptors. Understanding how nanovaccines elicit clonal bursts and somatic hypermutation needs to be addressed for the design of improved nanovaccines against highly variable viruses such as SARS-CoV-2 and influenza, where the success of immunization depends on eliciting extensive somatic hypermutation in antibody-secreting B cells.Outstanding questionsCan nanoscale materials be used to facilitate vaccine development?How do nanoscale properties such as size, shape, geometry, and surface functionalization contribute toward an effective immune response?How do nanovaccines complement the vaccine development process in the current pandemic scenario?Is it possible to acquire and track indicators of the long-term impact of nanovaccines over the lifetime of an individual?Alt-text: Outstanding questionsFinally, the promise of nanovaccines does not end with the simple induction of humoral or cell-mediated immunity, and nanovaccines represent a new frontier in the development personalized vaccines . However


Influenza B virus (IBV) is one of the two major types of influenza viruses that circulate each year. Unlike influenza A viruses, IBV does not harbor pandemic potential due to its lack of historical circulation in non-human hosts. Many studies and reviews have highlighted important factors for host determination of influenza A viruses. However, much less is known about the factors driving IBV replication in humans. We hypothesize that similar factors influence the host restriction of IBV. Here, we compile and review the current understanding of host factors crucial for the various stages of the IBV viral replication cycle. While we discovered the research in this area of IBV is limited, we review known host factors that may indicate possible host restriction of IBV to humans. These factors include the IBV hemagglutinin (HA) protein, host nuclear factors, and viral immune evasion proteins. Our review frames the current understanding of IBV adaptations to replication in humans. However, this review is limited by the amount of research previously completed on IBV host determinants and would benefit from additional future research in this area. Severe influenza infections in humans are typically caused by two influenza virus types: influenza A viruses (IAV) and influenza B viruses (IBV). These infections are suspected to cause up to 5 million severe cases of influenza disease annually . In the Throughout recorded history, IAV has been responsible for four major pandemic outbreaks, in 1918, 1957, 1968, and 2009. These pandemics were caused by the H1N1, H2N2, H3N2, and H1N1 subtypes, respectively . The strDue to shared structural and replicative properties with IAV, we hypothesized that the distinct difference in host range between IBV and IAV is unlikely to be due to chance alone. Viral and host factors important to IAV host determination have been reviewed previously ,14. HoweIn the initial stages of infection, IBV must bind to sialic acid residues on the surface of the cells lining the respiratory tract. Receptor binding is facilitated by the IBV hemagglutinin protein (B-HA). This receptor-binding interaction is crucial for successful viral infection and plays a major role in host specificity of IAV ,16,17. TTo facilitate membrane fusion between the viral envelope and the host endosome after receptor interaction, B-HA must have been enzymatically activated . While oOutside of receptor binding and enzymatic activation, Laporte, et al. has shown that the physical environment of the human upper respiratory tract plays a key role in facilitating IBV infection. Interestingly, the pH required for efficient B-HA activation and membrane fusion was lower for IBV strains than for avian IAV (5.4–5.6 for IBV compared to 5.7–5.8 for avian IAV) . AdditioThe receptor abundance in the human respiratory tract, the presence of enzymes capable of B-HA activation, and the optimal physical environment for IBV infection suggest possible host restriction of IBV replication to humans over non-human hosts. A summary of these factors can be seen in Influenza viruses belong to a group of RNA viruses that replicate their genome in the nucleus rather than the cytoplasm ,48 despiOnce in the nucleus, B-Pol transcribes the viral RNA to initiate the viral replication cycle. Human acidic nuclear phosphoproteins (ANP32) are crucial for influenza RdRp activity ,55,56,57Again, few studies describe the interactions of IBV with host nuclear factors for the replication process. As mentioned briefly, the interactions of IBV with human importin-α focus on binding affinities but do not investigate how non-human homologs impact viral replication. These studies also fail to fully establish a mechanism of nuclear transport across the nuclear envelope. To support importin-α acting as a host restriction factor, future studies should assess the ability of different species’ importin-α to support IBV replication. This would provide evidence regarding to what extent IBV can utilize other species’ importin-α for nuclear transport to replicate the viral genome and express the viral proteins. While the importance of human ANP32 proteins was shown more clearly for IBV replication than importin-α, only avian ANP32 proteins were used to compare more than the two primary forms of ANP32 proteins. A major limitation to the studies showing ANP32 proteins supporting B-Pol activity was the absence of a comparison between the ability of host ANP32 proteins to support viral replication. Only B-Pol activity was measured in a minigenome reporter assay . AnotherA critical relationship between a host and pathogen is immune evasion. Influenza viruses specifically encode proteins to aid in immune evasion and utilize proteins with other viral functions to assist in evasion. A major difference between IAV and IBV in immune evasion is the lack of alternative polymerase protein products encoded in the PB1 and PA gene segments. IAV PB1-F2  and PA-XThe interferon (IFN) and interferon-stimulated gene (ISG) signaling pathways are a primary defense for the innate immune system in humans. During influenza infection, host cells produce IFN to activate host ISGs and limit total gene expression . This imIn addition to avoiding innate immune detection within the cell, the virus must also evade any adaptive immunity to infected cells. This recognition can come from cytotoxic T lymphocytes (CTLs). One way the virus attempts to avoid CTL recognition is through the downregulation of MHC Class I surface expression in infected cells. After IBV infection, cell lines stably expressing different HLA-alleles showed significantly decreased MHC Class I surface expression compared to IAV infection . AnotherWe hypothesized that a virus with a limited host range such as IBV will be effective at evading its primary host’s immunity but not other host immunities. This would be due to the absence of viral responses to immune pressures exerted by non-human hosts, limiting virus replication. We also hypothesize that a virus with multiple mechanisms of immune evasion is more likely to have an expanded host range. The literature characterizing the B-NS1 interaction with human ISG15 supports our first hypothesis. As described above, B-NS1 is only able to rescue virus replication in the presence of human ISG15, while ISG15s from non-human species are able to inhibit replication in the presence of B-NS1. This suggests that IBV could be unable to successfully evade an aspect of non-human host immunity that prevents viral replication and spread within that host. We expect this species-specific targeting of ISG15, combined with other immune evasion mechanisms, plays a key role in promoting IBV circulation in humans while also potentially restricting replication in other non-human hosts.Other immune evasion mechanisms have been characterized for IBV. Protein kinase R (PKR) ,88, humaThe lack of an established non-human host presents a major difference between influenza B viruses and influenza A viruses. With the exception of isolated mammalian detection , no majoThe most extensively interrogated, possible IBV host restriction factor is the B-HA protein. The interaction between viral protein and host receptor is a crucial determinant of viral entry into susceptible cells. Unsurprisingly, host receptor characteristics likely shape whether or not that host can be infected. These characteristics are summarized in Possible host restriction factors outside of B-HA are not as well understood. To determine if a host restriction phenomenon has been observed during IBV genome replication, we considered host nuclear factors. The importin-α protein family has been identified as a critical factor for transporting the vRNPs from the cytoplasm into the nucleus ,52. HoweIBV has also evolved several mechanisms to escape human immune recognition. B-NS1 drives multiple mechanisms that obstruct the human IFN response ,74. TargA major theme to this research topic is the relative lack of studies investigating the possible IBV host restriction factors. Numerous studies have identified host factors or host–viral interactions critical for efficient IBV viral replication. However, these studies often fail to compare the human factors with non-human homologs, leading to a gap in our knowledge of why IBV only circulates in humans. Another potential avenue to discovery of IBV host restriction factors is considering the restriction factors of other types of influenza viruses. Factors that limit IAV, especially avian IAV, have been comprehensively reviewed ,14,93,94Two other influenza types have also been discovered and circulate in mammals. Influenza C viruses (ICV) are distantly related to IBV and primarily infect humans . InfluenDespite an established host restriction environment for influenza viruses, host spillover events are detected. The most well-known examples are the pandemic spillover events from past influenza pandemics . HoweverA potential piece of non-molecular evidence supporting strong host restriction factors for IBV circulation is the possible extinction of the Yamagata-like lineage. The 2019–2020 flu season was predominantly characterized by the emergence of the new Victoria-like clade V1A.3 ,46. ThisBased on our review of the available literature, there appears to be some evidence of IBV adaptation to human circulation throughout different stages of the replication cycle. Researchers have identified crucial interactions during receptor binding, genome replication, and immune evasion between viral and human factors that permit optimal virus replication to occur. The fact that these interactions occur throughout the virus replication cycle and not just during viral entry suggests that there is likely an important relationship between the human host and IBV replication. Many of these interactions require more comparison studies to investigate the impact that the factors from different host species have on IBV replication. This would help support the role of these factors as IBV host restriction factors. However, logically analyzing the available evidence suggests these adaptations could limit the ability of IBV to circulate in hon-human hosts. A better understanding of these interactions could unlock new information about how influenza viruses are restricted to their hosts. This information could then be leveraged as novel, specific antiviral targets in the future.