9: Interferons

Interferons (IFNs) Overview

• Interferons are cytokines with antiviral and growth-inhibitory properties
• Cytokines are the first line of defense against viral infections
• These have key roles in immunosurveillance against malignant cells

Classes of Interferons

• There are 3 classes of IFNs that include related cytokines:
• Subdivided into 13 subtypes.
• Type I IFNs include IFN-α, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, and IFN-ω1–3 and all have considerable structural homology
• Type 2 IFNs mainly consist of IFN-γ
• Type 3 IFNs mainly consist of IFN-λ (IFN-λ1, -λ2, -λ3) or interleukin-29 (IL-29), IL-28A, and IL-28B

Interferon Receptors

• Type I and Type II IFN receptors have multichain structures, composed of at least two distinct subunits
• Type I IFN receptors: IFNAR1 and IFNAR2
• Type II IFN receptors: IFNGR1 and IFNGR2
• Each receptor subunit interacts with a Janus activated kinase (JAK) family member
• Type I IFN receptor: IFNAR1 subunit associates with tyrosine kinase 2 (TYK2), while IFNAR2 associates with JAK1
• Type II IFN receptor: IFNGR1 subunit associates with JAK1, and IFNGR2 associates with JAK2

IFN-α/Type I IFN Binding

• The binding of IFN-α or other type I IFNs to the type I IFN receptor leads to rapid autophosphorylation
• This binding activates the receptor-associated JAKs TYK2 and JAK1
• This in turn regulates STATs phosphorylation and activation

STAT Activation

• Activated in response to type I IFNs: STAT1, STAT2, STAT3, and STAT5
• Activation is a common response to different type I IFNs, as they bind to the same receptor
• This in turn activates a common pathway involving the same JAKs such as TYK2 and JAK1
• IFN-α can also activate STAT4 and STAT6, but is restricted to endothelial cells or cells of lymphoid origin

STATs Post-Phosphorylation

• Activated STATs form homodimers or heterodimers after phosphorylation by JAKs
• These then translocate to the nucleus and initiate transcription
• It does this by binding to specific sites in the promoters of IFN-stimulated genes (ISGs)

ISG Factor 3 (ISGF3) Complex

• An important transcriptional complex induced by Type 1 IFNs
• The mature ISGF3 complex contains phosphorylated STAT1, STAT2, and IRF9 which does not undergo tyrosine phosphorylation
• This complex binds IFN-stimulated response elements (ISREs) in promoters of ISGs

Other STAT Complexes

• Other STAT complexes induced by type 1 or 2 IFNs: STAT1-STAT1, STAT3–STAT3, STAT4–STAT4, STAT5–STAT5, and STAT6–STAT6 homodimers
• Others still include: STAT1-STAT2, STAT1-STAT3, STAT1-STAT4, STAT1-STAT5, STAT2-STAT3, and STAT5–STAT6 heterodimers
• IFN type 2 induced complexes bind to the IFN-γ-activated site (GAS) in the promoter of ISGs
• Known Interferon-stimulated genes can contains either ISREs or GASs by themselves or both

CRKL and STAT5 in IFN Signaling

• CRKL constitutively associates with guanine-nucleotide-exchange factor (GEF) C3G, and is in a latent cytoplasmic form
• STAT5, is associated with with tyrosine kinase 2 (TYK2), which is bound to the type I interferon (IFN) receptor subunit IFNAR1
• After type I IFN receptor engagement by IFN, CRKL rapidly is phosphyorylated
• The activated CRKL forms signalling complex with STAT5, also undergoing TYK2-dependent tyrosine phosphorylation
• The CRKL-STAT5 complex translocates to the nucleus and binds to specific GAS in the promoters of certain IFN-stimulated genes (ISGs) Induction:
• CRKL also induces GEF activity of C3G
• C3G regulates guanine-nucleotide exchange small G-protein RAP1
• This is resulting in G-protein RAP1 activating and promoting growth-inhibitory responses

Organoids and SARS-CoV-2 Infection

• SARS-CoV-2 infects human airway organoids
• SARS-CoV-2 infects human small intestinal organoids
• Expands when sheared and exposed to virus at MOI = 1, roughly 100,000 cells/well
• "Inside" of organoids corresponds to the intestinal lumen
• “Outside” to the rest of the body. Immunofluorescent Imaging of Infected Organoids:
• SARS-CoV-2 infects dividing and mitotically inactive cells
• Ki67 indicates dividing cells APOA1 indicates post-mitotic cells

ACE2 and SARS-CoV-2

• The expression of ACE2 is almost non-existent in expanding organoids
• despite them still having the highest rate of SARS-CoV-2 infection
• ACE2 expression increases 600-fold upon differentiation, according to qPCR analysis

ACE2 Distribution in Airways

• The single-cell RNA sequencing revealed ACE2 is concentrated in the nasal pathway
• the lower airway, however, contains little ACE2 protein

SARS-CoV2 and SARS

• SARS-CoV2 and SARS can also induce innate immune responses in organoids