Abstract:
Microbiota is basically an ecological community comprised of commensal, pathogenic and symbiotic microorganisms providing protection against incoming bacterial pathogens. They play a dominant role in modulation of host health by contributing to immunologic, hormonal and metabolic homeostasis. The immune system-microbiota interactions have a symbiotic relationship where microbiota plays an important role in the induction, training, and function of the host immune system and the immune system, in return, maintain these highly diverse and evolving microbes of the host. The immune system is dependent on the microbiota for protection against invading pathogens, but any disturbance in their close relationship can leads to significant risk of microbiota contributing to the disease. From recent studies, we can say that there is sound evidence about microbiota providing protection against viral and autoimmune diseases but also in some diseases they promote the disease progression. Evaluation of complete environment that underlines a disease is extremely important as we move forward in our research and therefore the purpose of this review article is to summarize microbiota’s mechanism in the immune system, evidence of microbiota as immunity booster in different viral diseases, summarizing recent clinical trials of microbiota on different viral diseases and to suggest future directions on research strategies on interaction between immune system and microbiota.
Introduction:
Multicellular organisms are composed of the macroscopic host and its symbiotic commensal microbiota. These complex communities of microbes include bacteria, fungi, viruses, other microbial & eukaryotic species.1,2 The immune system-microbiota interactions have a symbiotic relationship where microbiota plays an important role in the induction, training, and function of the host immune system and the immune system, in return, maintain these highly diverse and evolving microbes of the host.3 This symbiotic interaction between microbiota and the host helps in functioning of immune system in mammalians, plants, insects and aquatic organisms. Furthermore, the immune system and microbiota also engage “cross-talk” which involves exchange of chemical signals in many animals. This helps immune system to recognize the types of bacteria which are harmful to the host and then combating them, and it also helps in identifying the beneficial microbiota and allowing them to carry out their functions like immune reactivity and targeting.4-5
Mechanisms of microbiota in the immune system:
Cytokine induction:
Microbiota modulates immune response by acting locally and systemically. Shift in composition and density of microbiota affects systemic immunity. It promotes dermal T cell function and thereby producing IL-1α which in turn directly controls the capacity of dermal resident T cells in to producing inflammatory cytokines like IFN-γ and IL-17A. Furthermore, oral cavity also harbors a unique and complex microbiota which have role in promoting the inflammasome activity resulting in local increase of the inflammatory cytokine IL1β.6Reduction of gut microbiota via broad-spectrum antibiotic treatment have shown blunted T and B cell response against intranasal infection with influenza which helps in considering its role in promoting the inflammasome-mediated induction of IL-1β and IL-18 secretion.7-8
Induction of regulatory responses:
Tissue homeostatsis maintenance is extremely important for the host survival and it depends on a complex and coordinated set of innate and adaptive responses involving microbiota and pathogens. Foxp3 regulatory T (Treg) cells helps in maintaining both peripheral and mucosal homeostasis throughout the lifespan of the host.9-11 If disruption occurs in the homeostasis of these cells, it would cause loss of oral tolerance and development of aberrant effector responses in the gut. Induction of Treg cells is known to confer a health benefit to the host. Microbiota also helps in controlling oral antigen sampling by mucosal DCs and they also promote the induction of lamina propria resident macrophages by local expansion of Treg cells.12-13 Also, tissue specific factors like Vitamin A and MUC2 which are produced by intestinal goblet cells helps in regulation of mucosal dendritic cells.14-15Production of polysaccharide A (PSA) by a prominent human symbiont Bacteroides fragilis provided first demonstration of induction of regulatory responses by microbiota.16 B. Fragilis helped in promoting Treg cell function and induction via engaging the microbial derived PSA with TLR2 expressed by T cells.17Furthermore, SCFA and in particular butyrate also helps in regulating the size and function of the regulatory T cell network by promoting the induction of regulatory T cells in the colonic environment.18-20
Induction of protective responses:
Microbiota have the capacity to shift from mutualist to commensal to parasite depending on the state of activation of the host, co-infection or localizations. Microbiota induces protective immune response by different mechanism to modulate the immune response. Firstly, they compete for nutrients, produce antimicrobial molecules and metabolites that affect the survival and virulence of pathogens. Furthermore, they also Promote the production of antimicrobial peptides by epithelial cells and reinforce tight junctions that directly affect pathogen growth or survival. In addition, microbiota also modulate the function of dendritic cells and other innate cells both locally and systemically so that it promotes the induction of effector T and B cells responses against pathogens.21
Mucosal firewall:
Microbiota minimize contact between microorganisms and the epithelial cell surface by maintaining homeostatic relationship with the host which further limits tissue inflammation and microbial translocation. This segregation is accomplished by the using epithelial cells, mucus, IgA, antimicrobial peptides and immune cells which are collectively known as “Mucosal firewall”.22 Each component of “mucosal firewall” performs different functions. Firstly, mucus acts as a primary barrier and it limits the contact between the microbiota and host tissue and thereby prevents microbial translocation.23 Epithelial cells also play an important role in limiting exposure to the commensal microbiota by producing antimicrobial peptides.24Tissue resident macrophages helps in eliminating translocating microbiota. Limitation of Microbiota is also done by CD103+ CD11b+DCs congesting to the mLN from the lamina propria but they do not penetrate deep and it leads to differentiation of commensal specific regulatory cells (Treg) Th17 cells and IgA producing B cells. Microbiota specific lymphocytes also traffic to Peyer’s Patches where Treg can further promote class switching and IgA generation against commensals.25
Formation of feedback loops:
Microbiota influence innate immunity by forming feedback loops. This loops are being regulated by various layers of intestinal wall and they perform three steps for stimulating innate immunity. Firstly, they recognize pattern recognition receptors like Toll-like receptors, the nucleotide-binding oligomerization like receptors, the RIG-I-like receptors, the C-type lectin receptors, the absent in melanoma 2 like receptors and the OAS like receptors. Then they mediate transcriptional response of the host; and, finally secrete effector molecules to mediate the immune response. The advantage of using such confined feedback loops is that the inflammatory response can be limited to the epithelial layer as they do not involve entire tissues or multiple organs. This loops involve communication between epithelial, myeloid and lymphoid cells using cytokines such as IL-18, IL-22, IL-23 and chemokines for mediating immune response.26
Transcriptional reprogramming:
Microbiota influence the activity of the innate immune system by transcriptional reprogramming. Transcriptional reprogramming involves expression of genes that are involves the host nutrient absorption and processing, barrier functions, gut motility, intestinal immune responses, angiogenesis and the metabolism of xenobiotics. Constituents of the microbiota have been involved in the regulation of ubiquitin signaling, protein neddylation, the nuclear translocation of transcription factor and vesicle trafficking.27Hooper et al, in their study found that reprogramming of intestinal gene expression was done by animals that were colonized with a single commensal microbiota. Microbial colonization and transcriptional responses are in part evolutionarily conserved as from evidence of reciprocal transplantations between mice and zebrafish.28
Epigenetic programming:
Microbiota modulate the immune response by epigenetic programming. Analysis of epigenetic modifications in the intestinal epithelial cells of germ-free mice have shown reduction in level of methylation for the gene that encodes the lipopolysaccharide sensor TLR4, which shows that commensal bacteria might induce tolerance through the epigenetic repression of pattern recognition receptors. Also, colonization of microbiota in germ-free neonatal mice has shown reduction in the methylation level of the chemokine-encoding gene Cxcl16.29Furthermore, comparison of mononuclear phagocytes from colonized and germ-free mice have shown that the microbiota promotes the trimethylation of histone H3 at lysine 4 at the loci of inflammatory genes, including those which encode the type I interferons.30The acetylation of histones is extremely important for the crosstalk between the microbiota and the immune system. It has been shown that deletion of histone deacetylase 3 results in massive alteration of gene expression and loss of the epithelial barrier integrity.These aberrations are known to be dependent on microbiota because germ-free mice that lacked intestinal histone deacetylase 3 do not present the same phenotype as their colonized counterparts.31
Microbiota as an immunity booster in different viral diseases:
Rotavirus
From many years there are evidences about boosting effects of microbiota on rota virus.Rota virus,a double-stranded, nonenveloped RNA virus from the Reoviridae family, is significant cause of viral diarrhea worldwide.Microbiota have a strong influence on rota virus as they have been to shown reduce the duration of viral diarrhea.administration of Lactobacillus rhamnosus GG reduces rotavirus shedding.32-34Soluble factors from commensal microbiota boosts immunity against rotavirus by blocking it in in intestinal epithelial cells in vitro. Varyukina et al. in their study suggested that soluble factors modify the intestinal epithelial cell-surface glycans and prevent attachment of rotavirus.35
Influenza
Stimulation of the host immune system by the microbiota has been shown in case of influenza virus disease. Influenza virus, a negative-stranded enveloped RNA virus from the Orthomyxoviridae family which spreads by the respiratory route.Dolowy et al. examined influenza virus pathogenesis in conventional versus germ-free mice and concluded that germ-free mice are more susceptible to influenza A virus compared to conventional mice.36Ichinohe et al. in their study found higher pulmonary influenza virus titers in antibiotic-treated mice compared with conventional mice8 and also,stimulation with toll-like receptor (TLR) agonists was sufficient to restore immune responses in antibiotic-treated mice, indicating that certain gut bacteria may prime the immune system for influenza virus protection.Abt et al. demonstrated increase in influenza virus titers and pathogenesis in antibiotic-treated mice compared with conventional mice. Reduction in virus-specific CD8+ T cell responses and IgG and IgM antibody levels correlated with the enhanced viral replication and disease in antibiotic-treated mice which indicates weaken adaptive immune responses in mice with depleted microbiota. In addition, antibiotic treatment impaired antiviral immune responses in alveolar macrophages.37Recently, Wang et. al. found that M2 alveolar macrophages were downstream mediators of viral clearance after priming with Staphylococcus aureus,a upper respiratory tract commensal bacterium.38These macrophages were demonstrated reduction in influenza pathogenesis by limiting inflammation in the lung. Priming was TLR2 dependent which confirmed the role of pattern recognition receptors in stimulating influenza immune responses. From these findings we can suggest that microbiota from the intestinal and upper respiratory tracts may play important roles in limiting influenza virus infections by providing a necessary signal to the immune system.
LCMV:
LCMV is a negative-strand ,enveloped RNA virus from the Arenaviridae family which undergo acute or persistent infection in mice depending upon the viral strain.Abt et al. demonstrated that microbiota promote antiviral responses in antibiotic-treated mice as there was impaired viral control of LCMV correlating with reduced LCMV-specific CD8+ T cell responses and IgG antibody titers and also,CD8+ T cells from antibiotic-treated mice demonstrated increase in inhibitory receptors and dep production of effector molecules indicating that T cell exhaustion occurs in the absence of conventional microbiota.Futhermore, in antibiotic-treated mice macrophage recruitment was not impaired but,macrophages from conventional mice expressed higher antiviral response genes, indicating an impaired innate immune response in antibiotic-treated mice. From this information we can say that the altered environment in antibiotic-treated mice diminishes innate and adaptive immune responses to LCMV infection.39
Dengue:
Microbiota have shown boost in immunity against dengue virus, single-stranded enveloped RNA virus from the Flaviviridae family. Xi et al. in their study treated Aedes aegypti mosquitoes with antibiotics with a expectation of depleting the midgut microbiota and then they examined effects on dengue virus replication. They found microbiota limit viral replication as there was higher dengue virus loads in the midgut of antibiotic-treated mosquitoes.39-40 Knockdown of the toll-like receptor adapter protein MyD88 also have shown increase viral loads in the midgut, indicating that the toll pathway is involved in limiting dengue virus replication. It seems that microbiota is the likely source for toll pathway stimulation which culminates in antiviral defense. Infection of mosquitoes with Wolbachia species confers dengue virus resistance as it induces oxidative stress within the host mosquito that activates the antiviral toll pathway but still future work is still required to understand the exact mechanism by which Wolbachia effects on dengue virus replication.41-43
Adenovirus:
Adenovirus, double-stranded nonenveloped DNA virus from the Adenoviridae family also have shown evidences about boost in immunity from the microbiota. In response to microbiota adenovirus replication is inhibited by defensins ,which are antimicrobial peptides produced by the host.44Defensins limit replication in vitro by directly binding the adenovirus virions. Therefore, we can say that the microbiota may limit adenovirus infection through induction of host defense mechanisms.45
Coxsackie B virus & Friend virus infection
Microbiota have shown boost in immunity against Coxsackie B virus,a pathogenic enterovirus from Picornaviridae family and Friend virus, murine leukemia virus of Retroviridae family. Schaffer et al, and Mirand et al, have shown in their studies that germ-free mice have enhanced susceptibility to coxsackie B virus and Friend virus respectively. They both suggested that microbiota directly or indirectly limits disease in mice with a conventional flora.46-47
Hepatitis B virus infection:
There are various clinical experiments which found that intestional microbiota ameliorate liver damage in hepatitis B viral infection. Chou et al. found that the maturation of gut microbiota in adult mice stimulated liver immunity, resulting in rapid HBV clearance.48Xu et al, suggested that some individuals fail to react to hepatitis B vaccination as they are lacking some microbiota. So, we need to do further experimentation to find the corresponding bacteria absent in the microbiota so that we can solve the problem of why the hepatitis B vaccination did not work.49
Microbiota as an immunity buster in viral diseases:
Many researchers have demonstrated that microbiota can also act as an immunity buster in different diseases. Kuss et al, have shown that microbiota promote reovirus and polio virus infection based on their findings of reduction in replication and pathogenesis in antibiotic treated mice. They found that poliovirus-susceptible mice show lower mortality rates compared to untreated mice. Furthermore, they have shown that replication of the virus in the intestine of mice was dependent on the microbiota because antibiotic-treated mice secrete poorly infectious virus.51This replication did not require live bacteria because of the bacterial surface polysaccharides like LPS and peptidoglycan (PG)provide the same effect on virus infectivity.52Kane et al, suggested that microbiota use bacterial LPS to enhance viral tolerance through IL-10 production which has an inhibitory effect on the mouse mammary tumor virus(MMTV). They found that MMTV utilizes the innate immune Toll-like receptor for inducing the tolerance and evading the antiviral immune response and it uses LPS to trigger the Toll-like receptor.53
Recent clinical trials of microbiota on viral diseases:
There are many clinical trials done to evaluate microbiota’s efficiency in different vaccines and diseases. Microbiota like Bifidobacterium longum bv. infantis CCUG 52486, Bifidobacterium lactis HN019, Lactobacillus rhamnosus GG (LGG), Lactobacillus paracasei, Lactobacillus plantarum were used in clinical trials to evaluate its efficiency in developing influenza vaccine.57,66,67Bifidobacterium Longum, Lactobacillus rhamnosus were used in developing Monovalent Hepatitis B, Hexavalent DT, Acellular pertussis vaccine. LGG (ATCC 52103), Bifidobacterium breve, Propionibacterium freudenreichii ssp. Shermanii were tested for developing Diphtheria, Tetanus and whole cell pertussis vaccine. Lactobacillus rhamnosus GG was also used in testing its efficiency in developing Tetanus, Haemophilus influenza type b (Hib) and pneumococcal conjugate vaccine (PCV7). Lactobacillus GG and Fructooligosaccharide mix were evaluated for developing oral salmonella vaccine.60,61Microbiota such as Lactobacillus Acidophilus, Bifidobacterum bifidum, Bifidobacterium longum, Bifidobacterium infantis were tested to develop vaccine for Mumps, measles, rubella, and varicella (MMRV).65 Lactobacillus rhamnosus GG, Lactobacillus acidophilus, Lactobacillus bulgaricus, Bifidobacterium animalis subsp. lactis, and Streptococcus thermophilus are used in clinical trials of HIV infection.72-74
Future Perspectives:
From the previous research studies we can say that microbiota either initiate innate immune signaling to inhibit or promote viral diseases or they interact with virion for alteration of infectivity or the host responses. Inherently complex nature of studies of microbiota-virus interaction, require using human viruses that are capable of replicating in an efficient way in standard mouse strains or to use rodent viruses. We can also use other experimental systems like mosquitoes, drosophila and primates as they have shown good potential.41,44,54 We also suggest use of ex vivo and in vitro experiments in case of unavailability of animal model. Furthermore, understanding the ways by which microbiota influence viral diseases is much difficult.We need a careful approach during implementation of altering the host microbiota because of possibility of adverse effects on the host is more superior than viral infection itself. Therefore, in depth understanding mechanisms of specific microbiota-virus would be more pertinent and further clinical trials would provide an efficient way to assess the interaction between microbiota and immune system.52-56,70