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The 3rd International Medical Congress of Prof. Alireza Yalda Foundation in Medical Sciences

17-20th November 2012Tehran

The Role of Cytokine Storm in Influenza Pathogenesis

Presented by :Talat Mokhtari-Azad

Director of National Influenza Center WHO Collaborating Center

School of Public Health, Tehran University of Medical Sciences

Some people get horribly sick from the flu, and even die. Others just rest for a few days.

What’s behind this fateful variation?

How is it that the flu virus can have such a variable impact on people?

Nature 2011; 480 (8) S14

NATURE REVIEWS MICROBIOLOGY 2011; 595 (9)

Influenza A virus tropism

NATURE REVIEWS MICROBIOLOGY 2011; 595 (9)

Influenza A virus tropism

NATURE REVIEWS MICROBIOLOGY 2011; 595 (9)

Influenza A virus tropism

NATURE REVIEWS MICROBIOLOGY 2011; 595 (9)

Influenza A virus tropism

NATURE REVIEWS MICROBIOLOGY 2011; 595 (9)

PB1-F2 induces apoptosis, promotes bacterial growth and acts as interferon antagonist

HA determines receptor binding, and antigenicity and tropism depending on the presence or absence of a polybasic clavage site:

H5N1: PQRERRRKKR↓G H7N1: PEIPKR-RRR↓G

H1N1: PSIQ-------SR↓G H3N3: PEKQ-----TR↓G H2N2: PQIE------ SR↓G

NA promotes efficient release of viral progeny from infected cells

The genotype of PB1, PB2, PA and NP determine viral replication competence

NS1 is a multifunctional interferon antagonist

Viral factors

Viral and host factors influencing the pathogenesis of influenza A virus

NATURE REVIEWS MICROBIOLOGY 2011; 595 (9)

Understanding health conditions that increase suseptibility:

Asthma and COPD Cardiovascular disease

Obesity Diabetes

•Increased and sustained inflammatory responses contribute to disease severity

Host factors influence viral infection by direct or indirect interactions with viral proteins during different stages of replication:

Required factors include ATP6V0D1, CAMK2B, CLK1 and p27 restriction or regulatory factors

Understanding health conditions that increase suseptibility:

Elderly (>65 years of age) Children (< 2 years of age)

Pregnant women Immunocompromised individuals

Host factors

Viral and host factors influencing the pathogenesis of influenza A virus

NOVEL CONCEPT ABOUT INFLUENZA PATHOGENESIS

-The sequence of HA cleavage site is a major determinant of disease severity ( local versus systemic)

- PB1-F2 protein , which leading to apoptosis( e.g.1918/ H1N1 and in various isolates of H5N1), promotes and increases pathogenesis of secondary pneumonia infection

- PB1-F2 cooperate with NS1 to inhibit INF response

- NS1protein , which is an antagonist of INF-mediated antiviral host response and a modulator of adaptive immune responses , and can inhibit global host gene expression through its interaction with the nuclear cleavage and polyadenylation factors (cpsf4 or CPSF 30)

- NS1 contributes to the cytokine dysregulation that is seen in human that succumb to HPAI H5N1 virus infection.

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TISSUE TROPISM IN THE INFLUENZA PATHOGENESIS

- Influenza has different disease phenotypes most commonly URT, conjunctives, pneumonia, and encephalitis

- Seasonal flu has strong tropism to URT ( rhinitis, pharynitis, laryngitis), tracheo bronchitis

- HPAIV H5N1 weak tropism to URT and strong tropism to LRT which may explain in part its failure to transmit efficiently among human

- URT is lined by olfactory mucosa and HPAIV H5N1 replicate in olfactory mucosa of URL and virus spreads directly from OM to the olfactory bulb and rest of the CNS and causing severe meningitis, encephalitis

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TISSUE TROPISM IN THE INFLUENZA PATHOGENESIS

- H7N7 associated predominantly to conjunctivitis. And probably is mediated by a signal transduction pathway in corneal epithelial cells related to NF –kB

- HpH5N1 and Hp AIV H7N1 have strong tropism to LRT and attaches abundantly to Clara cells lining bronchioles, type II pneumocyte lining alveoli, and alveolar

- Macrophage in alveoli and this tissue tropism fit with the primary diagnosis in the acute stage by alveolar epithelial necrosis, flooding of alveolar lumina by edema fluid mixed with fibrin, erythrocytes, macrophages , and neutrophil and lining alveolar ducts by hyaline membranes

-H5N1 induced significantly higher production of the cytokine( TNF (in macrophages and may explain in part the severity of LTR disease and high fatality rate of H5N1 infection infection

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THE ROLE OF CYTOKINES IN THE IMMUNE SYSTEM• Pro-inflammatory cytokines, such as interleukin (IL)-1,

IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN) -γ, serve to recruit cells to the site of infection and mediate direct antiviral effects.

• The up-regulation of anti inflammatory cytokines, such as IL-10 and transforming growth factor (TGF)-β, also occurs during infection with the purpose of regulating the extent of inflammation.

• This forms of regulation are critical to contain excessive inflammation that can cause more harm than good.

• However, during some infections and pathological conditions, regulation fails, and an unbalanced cytokine response called a cytokine “storm” can develops, leading to uncontrolled inflammation and increased morbidity and/or mortality for the host.

Non-cytopathic virus Cytopathic virus Non-cytopathic virus

IFN-γTNF-αIL-10

IL-17IL-22IL-21

TNF-α

IL-4IL-5

IL-13

IL-21TGF-βIL-10IL-35

Almost all

Viruses HSVHCVHIV

HSVHCVHIV

Friend virusDengue virus

RSV

?

T cell cytokinesT cell cytokines

Virus inducersVirus inducers

Immunity Immunity Immunopathology Immunopathology

Infected tissue Infected tissue

Lymph nodeLymph node

TH1,TH2, TH17 cellsTH1,TH2, TH17 cells

Treg cellTreg cell

iDCiDC

pDCpDC

MQMQ

NeutrophilNeutrophil

IL-6IL-10

TGF-βIL-1β

IL-6IL-10

TGF-βIL-1β

B cellB cell

Treg cellTreg cell

CTLCTL

Type 1 IFNsType 1 IFNs

DAMPsPAMPs

DAMPsPAMPs

RNSIL-17ROS

IL-1β

RNSIL-17ROS

IL-1β

IFN-γTNF-α

Perforin Granzymes

IFN-γTNF-α

Perforin Granzymes

NK cellNK cell

Vasulitis glomerulonephritisVasulitis glomerulonephritis

Immune complex formation & complement activation

Immune complex formation & complement activation

Mature DCMature DC

CD4 T cellCD4 T cell

MHC-IIMHC-II

TCRTCR

IFN-γIL-12

IFN-γIL-12

IL-6 TGF-βIL-6 TGF-β -TGFβ TGF-β

IL-4IL-5

IL-4IL-5

IL-6 IL-21 IL-6 IL-21

TH1 TH1

TH17 TH17 Treg Treg TH2 TH2 TFH TFH

CTLCTLTH1,TH2& TH17 cells

TH1,TH2& TH17 cells

Tissue cellTissue cell

THE IMMUNE RESPONSE TO INFLUENZA

Production of proinflammatory cytokines by epithelial and immune cells increases vascular permeability, allowing the additional cells of the immune system to pass through the endothelial barrier and reach the infected tissue.

Continuous viral replication leads to a constant influx of immune cells into the site of infection and increased production of cytokines.

Without control mechanisms, this positive feedback loop between cytokines and immune cells results in hypercytokinemia, ultimately causing severe damage to the host.

HOST DETERMINANT OF PATHOGENESIS

-Highly PI viruses can induce the high plasma level of: INF gama- induced protein(IP10; also known asCXCL10)

-Monocyte chemoattractant protein 1( also known CCL2)

-Interleukin 8(IL-8), IL-6, IL-10 and other pro-inflammatory cytokines that correlate with an elevated pharyngeal viral load and increased frequency of death caused by viral pneumonia

-Increased and prolonged of transcription of type 1 IFN , inflammation and innate immune induction are seen during infection with HPH5N1 virus, resulting apoptosis of DC and severe pathology

-TNF and IL-1 increase infiltration level of leukocyte, neutrophil and macrophages

-CD8 effector T- cell, by producing IL-10 could control the lung inflammation and injury that is induced during acute influenza infection

-NK cell, B cell and antigen presenting cell are susceptible to influenza virus

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Pro-inflammatory mechanisms

Anti-inflammatory mechanismsAnti-inflammatory mechanisms

Cytokines and chemokines:IL-2, IFNs, TNF, IL-1β, IL-6, IL-8IL-12, IL-17, CCL2, CCL3 and CXCL2

Chemical mediators:Prostaglandins, histamines and cyclooxygenases

Reactive species:Reactive oxygen speciesReactive nitrogen species

Proteinases :Matrix metalloproteinases

Treg cells Cytokines: IL-10, TGF-β and IL-23

Chemical mediators:Resolvins, protectins, galectin 1 and galectin 9

Inhibitory receptors:PD-1, LAG3, TIM3 and CTLA4

Innate immune regulatory proteins :TLR signal inhibitors: A20, SOCS1 and SOCS3RLR regulators: ubiquitin ligaseNLR inhibitors: p202

Tissue damage Homeostasis Viral persistence

Pro-inflammatory mechanisms Anti-inflammatory mechanisms

Nature Reviews Immunology 2010; 10

Balance between pro- and anti-inflammatory mechanisms may decide the outcome of viral infection!

THE GOOD, THE BAD AND THE UGLY PATHOGENESIS IN INFLUENZA

The good : protection afforded by innate and adaptive immunity

- To activate rapid response recognition of RNA by toll like receptors and retinoic inducible gene I(RIG-1) is key

-These receptor leads to the production of proinfammatory cytokines and type I IFN-α and β

- These cytokines exert antiviral activity by inhibiting viral replication and jump stars the adaptive immune response (virus specific B and T responses) and result in clearance of the infection)

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THE GOOD

-Individual that have experienced an influenza infection in the past are more resistant to subsequent infection with influenza viruses of the same type (p2009 H1N1)

-Infection with influenza viruses also induce protective immunity to influenza A viruses of other subtypes ( heterotypic Immunity)

-Immune response to conserved proteins (NP, M) are at the basis of heterotypic immunity specially cross reactive T-cell response are involved.

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THE BAD

Increased virulence is often correlated with:

Increased virus replication Tropism Regulation of immune responseThe best known determinants of

virulence are the multibasic cleavage site of HA, receptor binding preference of HA, NA activity, PB2, PB1, NP, NS1

NS1 through it antagonise the innate response specially INF.

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THE UGLY

The excessive or dysbalanced immune response, especially in the LTR, may be determined

- Overproduction of cytokines such as TNF-α, IL-6, IL-8, IFN type I or II as well as chemokines can result in severe inflammation, including excessive recruitment of neutrophils and mononuclear cells at the site of infection. This may damage lung tissue , reduce respiratory capacity and cause severe disease and mortality (e.g. H5N1, 1918 H1N1)

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UGLY

-In sufficient expression anti-inflammatory molecule such as HLA-G, interleukin 10, transforming growth factor-beta could influence extent of inflammation and pathogenesis of infection.

-Importantly , alveolar macrophages expressing high level of CD200R are critical immune-suppressive cells that upon activation prevent elevated cytokine environment.

-In contrast to monocyte-derived macrophages that infiltrate the lung after infection starts, resident alveolar macrophages may be important in suppressing inflammation of the lung.

-Recently it was shown that activation of endothelial cells plays a central role in the initiation of excessive cytokine production and inflammatory cell recruitment.

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POSSIBLE THERAPEUTIC AVENUES Antiviral therapies that directly target the virus (including

NA inhibitors: oseltamivir and zanamivir, and M2 inhibitors: amantadine and rimantadine) are currently the main form of influenza treatment.

Viral resistance to M2 inhibitors is widespread among seasonal and pandemic influenza strains, while oseltamivir resistance is mainly observed in seasonal influenza and is not very common in pandemic influenza strains.

The success of oseltamivir and zanamivir lies in the fact that most influenza viruses have difficulty altering the NA site that they target, making viral escape difficult.

However, a low number of oseltamivir-resistant pandemic (H1N1) 2009 as well as H5N1 viruses have been reported in several countries.

POSSIBLE THERAPEUTIC AVENUES high rates of viral resistance to currently available drugs and

points to the need for strengthening available treatment options, including therapy that targets the host immune response.

Anti-inflammatory agents that dampen the cytokine responses during influenza infection have been shown to decrease morbidity and mortality .

There are a number of anti-inflammatory treatments used to treat autoimmunity, including TNF blockers, which may be of use in a cytokine storm situation during acute influenza infection.

Clearly, more research is required before many of these immunomodulatory concepts can move into the clinic.

CONCLUSION• Despite an increase in influenza literature over the last two

years, it is still unclear what causes the severe morbidity and mortality associated with pandemic influenza strains.

• One potential mechanism is the induction of a cytokine storm and an imbalance of protective versus pathogenic immune responses.

• Since cytokine storms are likely to arise through various mechanisms, treatment options should focus on the overall cytokine imbalance and associated immunopathology rather than targeting specific components of the storm.

• It is clear that more work is needed to fully understand the pathogenicity of pandemic influenza viruses and correlations of cytokine storms to disease severity, ongoing efforts in this area could lead to better therapeutic approaches to combat the next influenza pandemic.