Interferon-gamma and immune system

By Wat Mitthamsiri, MD.

Allergy and Clinical Immunology Fellow King Chulalongkorn Memorial Hospital

Interferon-Gamma

And

Immune System

Outline

• Introduction • What is interferon (IFN) • Classification of IFN • Interferon gamma (IFN-γ)

– History and biology – Roles with other immune components – Roles in infection defense – Roles in autoimmunity – Roles in allergy and hypersensitivity

INTRODUCTION

What is interferon (IFN)

• Interferons are proteins which produce antiviral and antiproliferative responses in cells.

• On the basis of their sequence interferons are classified into five groups: α, α-II (or omega), β, delta (or trophoblast) and γ.

• Except for γ-interferon, the sequence of all the others are related

PROSITE documentation PDOC00225, Swiss Institute of Bioinformatics, http://prosite.expasy.org/cgi-bin/prosite/prosite-search-ac?PDOC00225#ref1

InterPro: protein sequence analysis & classification, EMBL-EBI, http://www.ebi.ac.uk/interpro/entry/IPR000471

What is interferon (IFN)

• Roles of IFN: – Decrease tumor growth, inflammation, and

angiogenesis – Innate immunity (IFN-α and IFN-β) – Adaptive immunity (IFN-γ)

O Meyer, Joint Bone Spine 76 (2009) 464–473

Classification of IFN

• 3 main classes:

K M Pollard, et al., Discov Med, 2013, 16(87):123-131.


• Type I IFNs – Encoded by 17 nonallelic genes

• Lack introns • Located on chromosome 9 in humans

– Glycosylated proteins,160-200 amino acids – Sharing 30% to 55% homology

• Type II IFN – 140 amino acids and shares no homology

with type I IFNs



• Type III IFNs: 3 IFN molecules – IL-28A, IL-28B, and IL-29 – Co-produced with IFN-β – But act by binding to a different receptor from

type I IFN receptors



Presenter

Presentation Notes

Receptors of 3 types of IFN and signal transduction proteins. ISRE: IFN-stimulated response element; STAT: signal transducers and activators of transcription; ISGF3: IFN-stimulated gene factor 3, STAT1-STAT2-IRF9 complex; GAS: IFN-activated site; noyau: nucleus; cytoplasme: cytosol.

INTERFERON-GAMMA History and biology

Interferon-gamma (IFN-γ)

• Sole type II IFN • Made primarily by T cells and NK cells • More of an interleukin than an interferon?

– Modest antiviral activity – Prominent derivation from T lymphocytes – Wide-ranging functions

• Play roles in cellular and allergic immunity

J W Steinke, et al., Middleton’s Allergy 8th edition, 2013, 65-82.

IFN-γ: General history

A. Billiau, P. Matthys Cytokine & Growth Factor Reviews 20 (2009) 97–113

1965 Induction of an IFN activity in human PBMC by phytohemagglutinin

1966 Ag-specific induction of IFN activity during virus infections

1972 New IFN was named ‘immune IFN’

1973 IFN produced during DTH reactions named ‘Type II IFN’

1980 Nomenclature Committee: definitive name ‘IFN-γ’

1982 Dimeric structure of IFN- γ suggested, Cloning of IFN- γ from cDNA

Presenter

Presentation Notes

1972 IFN induced by anti-lymphocyte serum differs physicochemically from classical IFN! named ‘immune IFN’ 1973 IFN produced during DTH reactions differs physicochemically from classical IFN! named ‘Type II IFN’

IFN-γ: Structure

• Dimeric in solution • Each subunit

– 6 α-helices, that comprise 62% of the structure

– No β-sheet – Composed of 140 amino

acids – No homology with type I

IFNs S E. Ealick, et al., Science, New Series, Vol. 252, No. 5006 (May 3, 1991), pp. 698-702


IFN-γ: Sources

• During innate immune responses – Natural killer (NK) cells – Natural killer T (NKT) cells – Macrophages – Dendritic cells


IFN-γ: Sources

• In adaptive immunity – CD8+ T cells – Control of infection, – CD4+ T helper 1 (Th1) subset

• Promotes inflammatory responses • Clearance of intracellular pathogens • Class-switching to IgG2a, IgG2b, and IgG3


IFN-γ: Gene • Human chromosome 12 • Cytogenetic Location: 12q14 • Molecular Location on chromosome 12:

– Base pairs 68,154,769 to 68,159,740

National Library of Medicine (US). Genetics Home Reference [Internet]. Bethesda (MD): The Library; 2013 Sep 16 [cited 2014 April 3]. Available from: http://ghr.nlm.nih.gov/gene/IFNG.

IFN-γ: Expression regulation

• In innate immunity – IFN-γ production response to constitutive

expression of transcription factors • Eomes and T-bet (NK cells) • T-bet (NKT)

– These transcription factors bind to regulatory elements that are already accessible within the Ifng locus, leading to activation of Ifng transcription



• In adaptive immunity – Expression by CD4+and CD8+ T cells – Differentiation process of naïve CD4+ or

CD8+ T cells to Th1 or cytotoxic T effector cells requires:

• T cell receptor (TCR) stimulation • Multiple rounds of cell division • Induction of T-bet • Epigenetic modifications within the Ifng gene


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Presentation Notes

Epigenetic modifications within the Ifng gene that make regulatory elements accessible to T-bet and other transcription factors


• Epigenetic regulation: long non-coding RNAs (lncRNAs) Tmevpg1 (also known as NeST) – Positively contribute to IFN-γ production by

CD4+ and CD8+ T cells – Tmevpg1 is adjacent to the Ifng gene – Encoded on the DNA strand opposite to that

coding IFN-γ



• Epigenetic regulation: long non-coding RNAs (lncRNAs) Tmevpg1 (also known as NeST) – Tmevpg1 transcription is dependent upon

transcription factors, Stat4 and T-bet • Which also influence Ifng transcription in CD4+

Th1 T cells – Tmevpg1 transgenic mice:

• Increased IFN-γ • Immune to salmonella infection


IFN-γ: Signalling pathways

X Hu, et al., Immunity 31, October 16, 2009, 539-550.

Presenter

Presentation Notes

IFN-g-Induced STAT1 Activation Cycle Mediated by Tyrosine Phosphorylation and Lysine Acetylation. Unphosphorylated STAT1 dimers are present in the cytoplasm in an equilibrium state between a parallel and antiparallel configuration of STAT1 subunits. Upon activation of IFNGR signaling, STAT1 is phosphorylated by JAK kinases on tyrosine 701 and phosphorylation stabilizes a parallel dimer configuration that exhibits DNA binding activity. Phosphorylated STAT1 translocates to nucleus, binds to GAS DNA sequences, and activates transcription of STAT1 target genes. Active STAT1 in the nucleus undergoes acetylation on lysines 410 and 413, a process catalyzed by histone acetyltransferases (HATs) such as CBP. Acetylation flags STAT1 for dephosphorylation by the STAT1 phosphatase TCP45; an antiparallel dimer configuration facilitates efficient dephosphorylation and thus deactivation. Dephosphorylated STAT1 shuttles back to cytoplasm, where histone deacetylases (HDACs) such as HDAC3 and possibly Sirtuins deacetylate STAT1 and complete the phosphorylation- acetylation cycle. Deacetylation of STAT1 results in less efficient TCP45-mediated dephosphorylation and thus licenses STAT1 for IFNGRJAK-mediated tyrosine phosphorylation



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IFN-g Signaling Disrupts TLRInduced Feedback Inhibitory Loops



• IFN-γ enhances TLR-

induced TNF production by disrupting an IL-10-mediated inhibitory loop

• Increased activity of GSK3

• Negatively regulates IL-10 expression by suppressing activation of transcription factors CREB and AP-1

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IFN-g Signaling Disrupts TLRInduced Feedback Inhibitory Loops



• IFN-γ enhances TLR-induced

IL-6 and IL-12 production

• Disrupts inhibitory loop mediated by Notch target genes Hes1 and Hey1

• Downregulates intracellular NICD2 amounts

• Inhibits expression of Hes1 and Hey1

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IFN-g Signaling Disrupts TLRInduced Feedback Inhibitory Loops Hes1 and Hey1 are transcription repressors that negatively regulate IL-6 and IL-12 gene expression



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Signaling Mechanisms Associated with IFN-g-Mediated Attenuation of Tissue Destruction



• Via regulation of IL-1R and TLR signaling

• Inhibits IL-1 signaling in macrophages by downregulating IL-1RI expression

• Blocks induction of MMP downstream of TLR signaling by

• Superinduce transcription repressor ATF3 • Inhibit transcription activators CREB and

AP-1 • Inhibits CREB activity by suppressing its serine

phosphorylation • Inhibits AP-1 by downregulating nuclear protein

levels of its subunits

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• Inhibits osteoclastogenesis and

bone resorption

• Suppress expression and signal transduction of RANK, CSF-1R, and TREM2

• (Receptors critical for the process of osteoclastogenesis)

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• Attenuates fibrosis by:

• Suppresses TGF-βR signaling by

• Induction of inhibitory SMAD (SMAD7) • Direct inhibition of SMAD3 by STAT1

• Inhibits IL-4R signaling by induction of SOCS1

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Signaling Mechanisms Associated with IFN-g-Mediated Regulation of T Cell Differentiation and Function



• In Th1 cell differentiation

• IFN-γ-STAT1 signaling is critical for induction of T-bet and thus for sustaining the positive feedback loop

• Leads to heightened production of IFN-γ.

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• IFN-γ blocks Th2 cell differentiation

• By inhibiting IL-4-STAT6 signaling

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• IFN-γ and STAT1 can block Th17 cell differentiation

• Mechanism of action is not clear • Possibly suppresses Th17 cell by targeting

STAT3 (shown by dotted lines)

• Inhibit aryl hydrocarbon nuclear receptor (AHR)

• Suppression of TGF-β and IL-1 signaling by IFN-γ may contributes to inhibition of Th17 cell differentiation (not depicted)

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Presentation Notes

Signaling Mechanisms Associated with IFN-g-Mediated Regulation of T Cell Differentiation and Function Because STAT3 signaling from multiple cytokines including IL-6, IL-23, and IL-21 plays a pivotal role in mediating Th17 cell differentiation



• Regulates Treg cell differentiation and function.

• Block TGFβ-mediated Treg cell differentiation

• Upregulates expression of T-bet in Foxp3+ Treg cells

• Promotes expression of CXCR3 that regulates homing of T-bet+ FoxP3+ Treg cells to sites of Th1 cell inflammation

Presenter

Presentation Notes

Signaling Mechanisms Associated with IFN-g-Mediated Regulation of T Cell Differentiation and Function T-bet increases suppressive function of Treg cells T-bet+ FoxP3+ Treg cells effectively suppress Th1 cell inflammation in vivo

INTERFERON-GAMMA Roles with other immune components

IFN-γ and macrophages


1969 Lymphocyte-mediated activation of macrophagesMIF responsible?

1979 Several macrophage-activating factor (MAF) assays established

1983 Anti-IFN-γ antibody neutralizes MAF preparations

1985 Cloned IFN-γ possesses MAF activity


• Driving differentiation from inactive monocytes into potent effector M1 activated macrophages – Enhanced adherence, phagocytosis,

degranulation, and production of reactive oxygen and nitrogen molecules

• Responsible for their accumulation at the site of CMIR as cells newly capable of killing intracellular pathogens and cancers



• Activated M1 macrophages – Induced by IFN-γ – High producers of IL-1β, TNF-α, IL-6, IL-12,

and IL-23 – but not IL-10

– =>Proinflammatory and participate in Th1

polarization


IFN-γ and NK cells


1980 IFN-γ upregulates NK activity

1983 IL-2 may induce IFN-γ in NK cells

1993 Novel IFN-γ-coinducing factor IGIF/IL-18

1995 IL-18 induces IFN-γ in NK cells

1991 IL-12 induces IFN-γ in NK cells

IFN-γ and NK cells+PMN

• Stimulates killing by NK cells and neutrophils

• Stimulates adherence of leukocytes to endothelial cells through induction of ICAM-1


IFN-γ and APCs + DCs


1982 IFN-γ enhances MHC Class II expression in mononuclear phagocytes

1984 IFN-γ induces enzymatic breakdown of tryptophan

2000 IFN-γ optimizes IL-12 production by DCs IFN-γ-induced IDO conditions DCs to

become tolerogenic

1990 IFN-γ induces IDO in vivo

1996 IFN -γ enhances MHC Class II expression in DCs

Presenter

Presentation Notes

Indoleamino 2,3-dioxygenase (IDO), IDO is an immunomodulatory enzyme produced by some alternatively activated macrophages and other immunoregulatory cells (also used as an immune subversion strategy by many tumors). Interferon-gamma has an antiproliferative effect on many tumor cells and inhibits intracellular pathogens such as Toxoplasma and chlamydia, at least partly because of the induction of indoleamine 2,3-dioxygenase.

IFN-γ and APCs + DCs

• Directly stimulates Ag processing

• Stimulates antigen presentation via increased MHC class I and II expression

• Stimulates cytokine production


IFN-γ and Treg cells


1995 CD4+CD25+ Treg cells defined

2005 Defective functioning of Treg cells in IFN -γR KO mice with collagen-induced arthritis (CIA)

2006 IFN-γ can convert CD4+CD25 cells into CD4+ Treg cells able to suppress experimental autoimmune encephalomyelitis (EAE)

Release of IFN-γ by Treg cells

IFN-γ and T-helper cells


1973 T cell-replacing factor (TRF, T cell help for B cells) described

1977 Type II IFN inhibits antibody production in vitro

2005 Th17 cell lineage defined in mice IFN-γ inhibits differentiation of Th17 cells

and IL-17 production by activated Th memory cells

1984 IFN-γ proposed as a TRF

1988 Th1 and Th2 clones described Role of IFN-g in Th1/Th2 paradigm

INTERFERON-GAMMA Roles in infection defense

IFN-γ and infection defense

• Most important in vivo role • Establishing effective response towards

pathogens whose elimination from the body depends on phagocytosis and intracellular killing

• Weakly inhibits viral replication





Pathogen entry

NK cells produce IFN-γ primes mononuclear phagocytes for production of monokines:

TNF-a and IL-12

IFN-γ & TNF-a augment bacteriostatic potential of phagocytes

Guided by IL-12, Th1 response is mounted

Additional IFN-γ production by activated CD4+ and CD8+ T cells


• In Listeria infection, IFN-γ can: – Augment normal resistance – Restore compromised resistance – Rx with neutralizing Ab to IFN-γ abrogated

resistance – IFN-γ production during the first 2 days of

infection was critical for development of protective Ag-specific T cells



• In Mycobacterial infections – Application of IFN-γ on skin lesions of

lepromatous leprosy patients caused increased infiltration with lymphocytes and reduction in the local bacterial load

– Mice with a disrupted gene for the IFN-γ receptor were found to fail controlling infection with M. bovis.



• Mycobacterial infections in IFN-γ deficient mice – Unable to control sublethal doses M.

tuberculosis or M. bovis – Bacteria multiplied more extensively and

caused more widespread damage in affected tissues.

– Compromised in both innate resistance in early phase of infection, and also later development of protective immunity


IFN-γ and infection defence

• Important factor in directing the immune response towards the Th1 pathway

• Mitigates excessive extramedullary myelopoiesis

• Responsible for apoptosis of CD4+ T cells in the later phases of the immune response to mycobacteria

• Responsible for the appearance of ‘immunosuppressive’ macrophages


INTERFERON-GAMMA Roles in autoimmunity


IFN-γ and autoimmunity

• Mechanism is remain unclear • Epidermal transgenic expression of IFN-γ

leads to – Anti-dsDNA, – Anti-histone autoAb – Glomerulonephritis

• Transgenic IFN-γ expression in other sites does not lead to systemic autoimmunity??



• Possibly supporting evidence – Ability of IFN-γ to

• Promote B cell IgG class switching to more pathogenic autoAb

• Activation of IgG Fc receptors and complement • Contributes to disease severity

– In end organ damage, infiltration of IFN-γ secreting T cells resulting in macrophage activation, inflammation, and tissue damage


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Presentation Notes

More pathogenic Ab = IgG2a and IgG3 in mice


• Possibly supporting evidence – Mutated mice with reduced decay of IFN-γ

mRNA • Increased IFN-γ signaling and accumulation of

follicular helper T (Tfh) cells • Increased germinal center B cells and autoAb • IFN -γR-deficiency in these mice can prevent the

development of lupus



• Possibly contradict evidence – The experimental autoimmune diseases,

EAE, EAU and CIA, = Th17-driven – Conditions for optimal in vitro induction of

naive T cells differentiation into Th17 cells by IL-23 were found to include neutralization of endogenous IFN-γ


Presenter

Presentation Notes

Experimental auto-immune encephalomyelitis (EAE) experimental autoimmune uveitis (EAU) collagen-induced arthritis (CIA)


• Possibly contradict evidence – Ablation of IFN-γ resulted in increased

numbers of IL-17-producing T cells – Conclusion: Endogenous IFN-γ inhibits

differentiation of Th17 cells – Ablation of endogenous IFN-g should boost

disease in EAE and CIA.



• Possibly contradict evidence – Blocking of autoimmune diseases can be

done by injection of syngeneic Treg cells – In vitro treatment of CD4+CD25 cells with

IFN-γ cause their conversion into CD4+ Treg cells

– Evidence exists for induced Treg cells to rapidly release IFN-γ, that may be important for their suppressive activity




IFN-γ as a therapeutic target?

• Fontolizumab, – Humanized monoclonal Ab against IFN-γ – Showed some efficacy in patients with

Crohn’s disease – Phase II clinical trial investigating its use in

rheumatoid arthritis was terminated because the first phase did not meet the endpoint



• Amgen’s AMG 811 – Human monoclonal Ab – Being evaluated in safety trials with subjects

with DLE and subjects with SLE with and without glomerulonephritis



• Non-specific targeting of IFN-γ – Impact both innate and adaptive immunity – Deficiency of IFN-γ is associated with severe

infection • Targeting to cellular components

regulating IFN-γ expression, such as lncRNA Tmevpg1, may provide greater therapeutic benefit without adverse effect on responses to infection


INTERFERON-GAMMA Roles in allergy and hypersensitivity

IFN-γ and allergy

• Allergic inflammatory tissue has prominent presence of IFN-γ

• IFN-γ exacerbates allergic inflammation through its ability to – Activate accessory cell function – Stimulate cytokine secretion – Induce adhesion molecule expression – Activate eosinophils and neutrophils


IFN-γ and allergy

• IFN-γ promotes allergic inflammation – IFN-γ–producing Th1 lymphocytes exacerbate

murine asthma – Th1-like processes are particularly prevalent

in patients with severe asthma, especially those with irreversible obstruction and neutrophilic inflammation


IFN-γ and allergy

• It is frequently stated that the immune response to allergens in non-allergic subjects is characterized by Th1-like lymphocyte responses

• …But without CMIR and cellular inflammation


IFN-γ and DTH


• Exogenous IFN-γ was found to reverse inhibition of the DTH response by anti-CD4 or anti-IL-2R Ab in mouse model • supporting the concept that production of

IFN-γ by TH1 cells is essential for the reaction • There was report of IFN-γ potentiates

contact sensitivity

IFN-γ and DTH


• There were ambiguity in reports analyzing the role of IFN-γ in DTH reactions • This reflects the pathogenic complexity of the

systems under study • Effects may differ depending on • Ag used (protein or hapten) • Route of exposure (injection or contact with

the skin) • Time point during the reaction (during the

sensitization or the elicitation phase)

IFN-γ and DTH


• DTH reactions rely on both natural and acquired immune response mechanisms

• IFN-γ may act differently on these components

IFN-γ and Shwartzman reaction


• 2 varients: • Localized • Generalized

• Both are two-stage phenomena • Preparative (sensitizing) injection of

endotoxin • Eliciting (provoking) injection followed after

about 24 h

Presenter

Presentation Notes

Classically, to obtain a local Shwartzman reaction in the rabbit, the preparative dose is given in the skin, and the eliciting dose is given iv. Within a few hours a thrombohemorrhagic reaction appears at the prepared skin site. When both doses are given intravenously, a generalized reaction can occur, which often leads to death

IFN-γ and Shwartzman reaction


• Example of human model: • Thrombohemorrhagic shock that sometimes

occurs in humans with meningococcal sepsis • In mouse model with Pre-treatment with

neutralizing anti-IFN-γ • Completely protected against this reaction • Reduced production of circulating TNF

following the eliciting dose

TAKE HOME MESSAGE

Take home message

• IFN-γ is the only type II IFN • It’s a cytokine that is critical for innate and

adaptive immunity • Its action mainly via JAK-STAT pathway • It has multifaceted roles: Infection defense

(esp. intracellular pathogens) , CMIR, autoimmunity, allergy and hypersensitivity

Health & Medicine

Interferon-gamma and immune system