Host Immunity & Preserving the Healthy State

The body contains diverse biochemical mechanisms for preventing infection in the healthy individual. Influenza A viruses, including H1N1, infect the upper respiratory tract epithelium.1,2,3 The respiratory epithelium (with the exception of the alveoli) is composed of ciliated columnar pseudostratified cells coated in mucus, and plays an important role in protecting the body from a variety of pathogens. The airway becomes increasingly branched as it extends from the upper respiratory tract to the lower, beginning with the nasal passage and trachea, traveling to the bronchi and bronchioles, and then to the alveoli.4 

Figure 1. Anatomy of the human respiratory system. The airway becomes increasingly branched progressing from the nasal cavity to the trachea, bronchi, and alveoli in the lungs.23

Though exposed to a wide range of microbes from the air and environment, these epithelial cells possess powerful machinery in the healthy individual to prevent these pathogens from colonizing the respiratory tract.3,4 The non-alveolar ciliated epithelial tissues serve as an important physical barrier to infection, making it difficult for pathogens to travel quickly through the respiratory tract.4 A variety of immune cells also inhabit the respiratory epithelium, including dendritic cells, T-cells, lymphocytes, natural killer cells, and in the alveoli, resident alveolar macrophages.4 These regions also include goblet, serous, club, and neuroendocrine cells which secrete enzymes and other proteins essential to the immune response. 𝛽-defensin-I, for example, is constitutively expressed and secreted from human airway epithelia. This molecule binds the influenza virus and creates pores in its membrane to facilitate its destruction.15 𝛽-defensin has a vitamin-D response element in its promoter, which may contribute to the increased instance of influenza outbreaks during colder seasons. Mucins, an essential component of mucus, are often found tethered to these epithelial cells and act as a decoy, with structural similarity to the α-2,6 sialic acids which the influenza virus typically binds. The virus may bind this alternatively, preventing binding with the cell itself necessary for infection.4

On infection, a variety of innate immune receptors recognize signatures of viral infiltration. These include the toll-like-receptors (TLRs), retinoic acid-inducible gene-I (RIG-I), and nucleotide binding oligomerization domain (NOD)-like receptors (NLRs).24 Single stranded viral RNA is recognized by either TLRs 7 and 8 on the endosomal membrane or the cytosolic RIG-I receptor,24, 25 leading to type I interferon and pro-inflammatory cytokine production. This is mediated by interferon regulatory factor 7 (IRF7) or nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the TLR pathway, for IFN type I and pro-inflammatory cytokines respectively.25 Double stranded RNA is recognized by TLR3. The RIG-I pathway can also stimulate NF-κB, leading to production of type I IFNs.25 These proteins also encourage cytokine and chemokine production (IL-1β, IL-6, IL-8, TNFα, CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), and CXCL10 (IP-10))). CCL2 is implicated in macrophage recruitment to infected tissues.3

Figure 2. Graphical summary of the major innate immune components implicated in restricting influenza infection.24 Abbreviations: single stranded RNA (ssRNA), retinoic acid inducible gene I (RIG-I), inositol-3-phosphate synthase isozyme I (IPS-I), interferon regulatory factor 3 or 7 (IRF3 or IRF7), toll like receptor 7/8 (TLR 7/8), interleukin (IL), interferon (IFN), apoptosis associated speck-like protein (ASC), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), myeloid differentiation primary response protein (MyD88)

The interferons produced by these pathways act as either autocrine or paracrine signals which activate the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway.24, 25 Receptor activation leads to phosphorylation and dimerization of STAT1 and STAT2, which bind IRF9 and travel to the nucleus, where they upregulate the transcription of IFN-stimulated genes (ISGs).25

Influenza single stranded RNA is also recognized by the NLRP3 (member of the NOD-like-receptor family) inflammasome complex in the cytosol, implicated in caspase activity for interleukin activation.4,7 These immune molecules are received by endothelial cells, promoting chemokine formation and amplification, sometimes leading to a “cytokine storm” which can be harmful to the host when the immune response is too aggressive.7

Interestingly, some genetic factors can also play a role in the host immune response. A polymorphism of the IFITM3 gene is associated with increased susceptibility to influenza infection. The interferon-induced transmembrane protein 3 (IFITM3) protein product plays an important role in preserving the healthy state and preventing infection by directing viral components to lysosomes for degredation.8,9 Though IFITM3 is often discussed in the context of H1N1, this protein plays a role in preventing infection from all influenza A viruses. IFITM family proteins are widely conserved across eukaryotic species, and aid in preventing infection from not only Influenza A, but also the yellow fever virus, Marburg virus, Ebola virus, human immunodeficiency virus type 1 (HIV-1), and others.10 Influenza A viruses, though, are the primary target of IFITM3. Other members of the IFITM family are implicated in preventing infection from the aforementioned pathogens. The wildtype IFITM3 engages with viral particles and trafficks them to lysosomes for destruction.9 Cellular distribution of IFITM3 is controlled by phosphorylation of tyrosine residues,11 and the protein can’t retain its antiviral function without this post-translational modification.12 Other post-translational modifications, namely ubiquitination and S-palmitoylation diminish or increase the antiviral effects of this protein, respectively. The structure of IFITM3 is currently unknown, though it is thought to take on a single-transmembrane topology.14 Its localization to the membrane may have made structural study of this protein more difficult.

The mechanism of IFITM3’s antiviral function is not entirely understood.13 Interestingly, IFITM3 may exert its antiviral effect by altering cholesterol metabolism in the host cell. As discussed in question (2), cholesterol turns out to play an essential role in influenza entry, infection, and budding.14,15 In the presence of viral infection, IFITM3 interacts with vesicle-membrane-protein-associated protein A (VAPA), preventing VAPA association with oxysterol-binding protein (OSBP). VAPA-OSBP is essential for the transport of cholesterol to endosomes, organelles, and other cellular membranes. Thus, inhibition of this process by IFITM3 prevents this localization of cholesterol, interfering the the lipid rafts essential to viral function and replication, restricting infection.16 Interestingly, both IFITM3 and the Ch25h, the gene encoding cholesterol 25-hydroxylase (EC 1.14.99.38) which downregulates cholesterol synthesis and produces 25-hydroxycholesterol (25HC),26 are ISGs targeted by JAK-STAT pathway signaling.25 It is thought that increased cellular concentration of 25HC may also interfere with viral fusion.25

A mutagenic study of IFITM3 demonstrated that regions on both a conserved intracellular loop and transmembrane domain are essential for its function. F75 and F78 of the transmembrane domain were shown to mediate association of IFITM proteins, the loss of which reduced restriction of infection with the influenza A virus.17 A tyrosine on the N-terminal domain (Y20) was also identified as essential to proper localization of the IFITM3 protein. 

While innate immunity is important to restricting viral infection, the adaptive response plays a vital role as well. Both T- and B-lymphocytes serve important roles in adaptive immunity.25 Dendritic cells (DCs) residing beneath the respiratory epithelium can recognize and present viral antigens to nearby T-cells. These antigens are generated by DC mediated degradation of viral proteins to smaller peptide antigens, which are conjugated to major histocompatibility complex (MHC) proteins for presentation. Naive CD8+ T-cells activated by MHC-I on DCs translocate to the lymph nodes, where IFN and interleukin signaling promotes maturation to cytotoxic T-lymphocytes. Cytotoxic T-lymphocytes migrate back to the respiratory tract where they can secrete cytokines and other signaling molecules capable of inducing death in infected cells. CD4+ T-cells activated by MHC-II on DCs mature into helper T-cells, as well as Th1 CD4+ effector cells and regulatory cells. These cells secrete signaling molecules which stimulate B-cells and antibody production, as well as innate immune cells such as macrophages. Memory CD8+ T-cells will circulate in the blood post-infection.25

Figure 3. Graphical summary of the primary adaptive immune components.24 Abbreviations: T-cell receptor (TCR), activated cytotoxic t-cell (CTL), cluster of differentiation 4/8 (CD4/8).

Mature B-lymphocytes are associated with antibody production. Antibodies specific to HA are the most important in restricting viral infection. Neutralizing antibodies specific to the HA and NA proteins are secreted from mucosal tissues to prevent viral entry.24

Collectively, the innate and adaptive immune systems work together in preventing and restricting influenza infection in the healthy individual.

Summary of Major Scientific Innovations:

  • 1932-1933 – The influenza virus is identified and isolated20,21
  • 1933 – It is demonstrated that previous influenza infection prevents subsequent illness21
  • 1980s – Adaptive immune responses such as antibody protection and cytotoxic T-cell activity is investigated19
  • 2009 – IFITM proteins are first associated with restriction of influenza infection22
  • 2009 – NLRP3 inflammasome associated with restriction of influenza infection18
  • 1990s-present – Extensive mapping of infection mechanism and host immune pathways 5,12

Works Cited

  1. Krammer, F.; Smith, G. J. D.; Fouchier, R. A. M.; Peiris, M.; Kedzierska, K.; Doherty, P. C.; Palese, P.; Shaw, M. L.; Treanor, J.; Webster, R. G.; et al. Influenza. Nat. Rev. Dis. Primer 2018, 4 (1), 1–21. https://doi.org/10.1038/s41572-018-0002-y.
  2. Taubenberger, J.K. and Morens, D.M. The Pathology of Influenza Virus Infections. Annu Rev Pathol 2008 3:499-522. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504709/
  3. Fukuyama, S. and Kawaoka, Y. The pathogenesis of influenza virus infections: the contributions of virus and host factors. Curr Opin Immunol. 2012 23(4)481-486. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3163725/
  4. Denney, L. and Ho, L. The role of respiratory epithelium in host defence against influenza virus infection. Biomed J. 2018 41(4):218-233. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197993/
  5. Liu, J.; Stevens, D.J.; Haire, L.F.; Walker, P.A.; Coombs, P.J.; Russell, R.J.; Gamblin, S.J.; and Skehel, J.J. Structure of H1 1918 hemagglutinin with human receptor. PBD 2009 http://doi.org/10.2210/pdb2WRG/pdb 
  6. Javitt, G.; and Fass, D. Mucin 2 D3 domain. PBD 2019 http://doi.org/10.2210/pdb6RBF/pdb 
  7. Teijaro, J.R.; Walsh, K.B.; Rice, S.; Rosen, H.; and Micahel, B.A. Mapping the innate signaling cascade essential for cytokine storm during influenza virus infection. PNAS 2014, 111(10): 3799-3804. https://doi.org/10.1073/pnas.1400593111 
  8. Everitt, A.R.; Clare, S.; Pertel, T.; John, S.P.; Wash, R.S., Smith, S.E.; Chin, C.R.; Feeley, E.M.; Sims, J.S.; Adams, D.J.; Wise, H.M.; Kane, L.; Goulding, D.; Digard, P.; Anttila, V.; Baillie, J.K.; Walsh, T.S.; Hume, D.A.; Palotie, A.; Xue, Y.; Colonna, V.; Tyler-Smith, C.;  Dunning, J.; Gordon, S.B.; The GenISIS Investigators, The MOSAIC Investigators, Smyth, R.L.; Openshaw, P.J.; Dougan, G.; Brass, A.L.; Kellam, P. IFITM3 restricts the morbidity and mortality associated with influenza. Nature. 2012, 484, 519-523. https://doi.org/10.1038/nature10921 
  9. Spence, J.S.; He, R.; Hoffman, H.; Das, T.; Thinon, E.; Rice, C.M.; Peng, T.; Chandran, K.; and Hang, H. IFITM3 directly engages and shuttles incoming virus particles to lysosomes. Nature 2019 15, 259-268. https://www.nature.com/articles/s41589-018-0213-2
  10. Jia, R.; Pan, Q.; Ding, S.; Rong, L.; Liu, S.; Geng, Y.; Qiao, W.; and Liang, C. The N-Terminal Region of IFITM3 Modulates Its Antiviral Activity by Regulating IFITM3 Cellular Localization. J. Virology 2012 86 (24): 13697-13707. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3503121/
  11. Chesarino, N.M.; McMichael, T.M.; Hach, J.C.; and Yount, J.S. Phosphorylation of the Antiviral Protein Interferon-inducible Transmembrane Protein 3 (IFITM3) Dually Regulates Its Endocytosis and Ubiquitination. J. Biol Chem 2014 https://www.jbc.org/content/289/17/11986?ijkey=420e0f69937f1db9fc4d787fbe9130e7451647d0&keytype2=tf_ipsecsha
  12. Wellington, D.; Laurenson-Schafer, H.; Abdel-Haq, A.; and Dong, T. IFITM3: How genetics influence influenza infection demographically. Biomedical Journal 2019 42 (1): 19-26. 10.1016/j.bj.2019.01.004 
  13. Ling, S.; Zhang, C.; Wang, W.; Cai, X.; Yu, L.; Wu, F.; Zhang, L.; and Tian, C. Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3. Nature Sci Reports. 2016. 6, 24029. https://doi-org.muhlenberg.idm.oclc.org/10.1038/srep24029 
  14. Mañes, S; del Real, G.; and Martinez, A. Pathogens: raft hijackers. Nature Rev. Immunology 2003, 3, 557-568. https://doi.org/10.1038/nri1129 
  15. Schroeder, C. Cholesterol-binding viral proteins in virus entry and morphogenesis. Subcell Biochem. 2010, 51:77-108. doi: 10.1007/978-90-481-8622-8_3 
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  17. John, S.P.; Chin, C.R.; Perreira, J.M. et al. The CD225 Domain of IFITM3 Is Required for both IFITM Protein Association and Inhibition of Influenza A Virus and Dengue Virus Replication. J. Virol. 2013, 87(14); 7837-7852. https://dx.doi.org/10.1128%2FJVI.00481-13 
  18. Allen, I.C.; Scull, M.A.; Moore, C.B.; Holl, E.K.; McElvania-TeKippe E.; Taxman, D.J.; Guthrie, E.H.; Pickles, R.J.; and Ting, J.P. The NLRP3 Inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral RNA. Immunity 2009, 30(4):555-565. https://dx.doi.org/10.1016%2Fj.immuni.2009.02.005 
  19. McMichael, A.J., Gotch, F.M., Noble, G.R., Beare, P.A. Cytotoxic T-cell immunity to influenza. N Engl J Med. 1983, 309(1):13-17. https://www.nejm.org/doi/full/10.1056/NEJM198307073090103?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%3dpubmed
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  21. Shope, R.E. Studies on Immunity to Swine Influenza. J Exp Med. 1932, 56(4):575-85.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2132181/ 
  22. Brass, A.L.; Huang, I.C.; Benita Y.; et al. The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell 2009, 139(7):1243-54. doi: 0.1016/j.cell.2009.12.017
  23. Brain Kart. Anatomy of the Respiratory System (Image). http://www.brainkart.com/article/Anatomy-of-the-Respiratory-System_21909/ (Accessed Apr 14, 2020)
  24. Bahadoran, A.; Lee, S.H.; Wang, S.M.; Manikam, R.; Rajarajeswaran, J.; Raju, C.S.; and Sekaran, S.D. Immune Responses to Influenza Virus and Its Correlation to Age and Inherited Factors. Front Microbiol. 2016, 7:1841. https://doi.org/10.3389/fmicb.2016.01841
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  26. KEGG. Enzyme 1.14.99.38 (cholesterol 25-hydroxylase). https://www.kegg.jp/dbget-bin/www_bget?ec:1.14.99.38 (Accessed Apr 20, 2020).

For more information and resources, see Annotated Bibliography

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