Overproduction of Th1 and Th17 Cytokines may be the Clue to why some H1N1 Patients get very ill

18 12 2009

ResearchBlogging.org The present H1N1 influenza virus (nvH1N1, nv=new variant) behaves very differently from other influenza strains. The majority of nvH1N1 infections are mild and self-limiting in nature, but a small percentage of the patients require hospitalization and sometimes emergency care. Unlike the seasonal flu virus, the people who seem to suffer serious complications from this flu are not those over age 65, but for instance pregnant women, children, obese people, asthma patients and immunocompromised patients.

One explanation for the heterogeneous response may be that some groups of patients are extra vulnerable to flu-related complications, like pneumonia. (i.e. in asthma and pregnancy). It is also conceivable that some people respond differently to the H1N1 virus.  A new study published in Critical Care [1] may provide the first potential immunological clue of why some people will develop severe pneumonia when infected by the pandemic H1N1 virus.

People that needed hospitalization and critical care had a different pattern of cytokines (signaling molecules) in the blood compared to each other and compared to outpatients. The pattern of lymphokines (cytokines produced by lymphocytes) is typical for the different groups of T helper cells that produce them.

T helper cells
are T lymphocytes that assist B lymphocytes to mount an antibody response to viruses or other foreign bodies. There are different subtypes of helper cells, Th1, Th2 and the recently discovered Th17 cells [2]. These cells are required for protection, but act as a double-edged sword: Th2 have been implicated in asthma, and  Th1 and Th17 cells in many auto-immune diseases.  Interestingly, with respect to the present study, transgenic mice expressing IL-17 in lung epithelial cells showed substantial pulmonary pathology.[3]

How was this study done?
Both hospitalized and outpatients were recruited during the first pandemic wave in the months of July and August 2009 in ten different hospitals within the National Public Health System of Spain. Exclusion criteria were: bacterial infection, pregnancy, age (<16 yrs or >80 yrs) and patients whose samples were taken later than 5 days after hospital admission.

The following groups were studied:

  1. 15 control subjects (healthy donors)
  2. 15 outpatients (patients with mild, exclusively flu-like, symptoms)
  3. 10 hospitalized, non-critically ill patients
  4. 10 hospitalized patients, admitted to the intensive care unit due to respiratory insufficiency (these patients were slightly older than the other hospitalized patients); 3 patients ultimately died

Serum was analyzed for 30 different cytokines (and other mediators) and for determining viral load. Nasopharyngeal swabs were  taken for viral diagnosis.

Comparisons between groups were performed using the non parametric U-Mann Whitney test.

Results & Authors’ Conclusions:

  1. Increased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1β), and the absence of anti-nvH1N1 antibodies characterized the early response to nvH1N1 infection in all patients, whether hospitalized or not.
  2. High systemic levels of type-II interferon (IFN-γ) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17,IL-6) and T-helper 1 (TNF-α, IL-15, IL-12p70) responses were exclusively found in hospitalized patients.
  3. IL-15, IL-12p70, IL-6 constituted a hallmark of critical illness.

The diagram below illustrates the authors’ conclusion.


Are the conclusions supported by the data?

Conclusions of the authors
The authors use the non parametric U-Mann Whitney test to compare all H1N1 infected groups to the control groups. Thus the conclusions seem justified as far as H1N1 groups (2, 3, 4) are compared to controls (1) and not to each other. It is correct to conclude that groups 2, 3, 4 have more inflammatory cytokines than controls, but not to make the subdivisions as they did in the diagram and in the supplemented table.
It also not justified to conclude that IL-6 is the hallmark of critically ill patients”, as the authors did, because all nvH1N1-infected groups have higher levels of IL-6 versus control.
If you make more comparisons (30 cytokines) and compare more groups, you will definitively find more “significant results” by chance, not because the values are truly different (see Graphpad). Therefore more rigid statistical tests are needed. (Anova?)

Fig 2 from the article, adapted to show 4 out of 9 graphs

Conclusions of the media (and a Canadian co-author)
It is really surprising that all news articles I’ve seen on the subject report that levels of IL-17 in the blood appear to be culprit of causing severe symptoms in H1N1 infection (see Science Daily, News of the University of Toronto, Science Blog, Health Zone Canada). Weeks Update even produced the sensational headline: “Interleukin 17 (IL-17) responsible for death due to swine H1N1 flu”*

IL-17 is just one of the cytokines that is overexpressed in the present study. And -according to the above diagram-, it is less discriminative than IL-15 or IL-6. If we had to put the blame on something, it would be the Th1 and Th17 cells together, precisely as the title of the article indicates.

Perhaps the journalists mix up IL-17 with Th-17? But then, the Th1 subset producing TNF-α, IL-15, IL-12p70 seems more specific for critically ill patients than the Th-17 cells (at least on basis of the diagram).

Most news articles seem to be based on an interview with the Canadian senior study-author, dr Kelvin, who’s group did most of the IL-17 lab work. Excerpt from Health Zone Canada citing dr Kelvin and ted Ross:

The molecule, known as interleukin 17, may be the culprit causing severe symptoms in a host of seasonal influenzas and other respiratory ailments like SARS, he said.

It is this inflammatory function that is likely at play in flu sufferers with elevated levels, he said. “When we found that there were high levels of (interleukin 17) in severe patients and not in the mild patients, we thought that was a clue to what was driving the inflammation in the lungs of those severe patients.”

“If it’s high early in infection, it looks like you’re going to have a more severe outcome and therefore you can screen patients,” said Ross, in commenting on the paper.

Ross said interleukin 17 also presents a promising target for new drugs that could help patients recover from H1N1 pneumonias.

It seems that either the investigators know more than is published in the current paper or they base their ideas on what is already known in other infectious diseases and animal models. At any rate, these conclusions are not supported by the Spanish data. One look at the IL-17 results (Fig 2) makes it clear that -on basis of the current results- IL-17 can never be the sole predictor of a a severe outcome of H1N1 infection. It is also not known if IL-17 is a causative factor and if so, the only one. Therefore it seems much too early to conclude that IL-17 might be used to ‘screen’ patients and/or as a treatment modality.

* the authors even wrote: “The patient who died five days after disease onset showed high viral load and undetectable IL-17 levels in serum. This could reflect a protective role of IL-17 in severe patients.”


  1. Bermejo-Martin, J., Ortiz de Lejarazu, R., Pumarola, T., Rello, J., Almansa, R., Ramirez, P., Martin-Loeches, I., Varillas, D., Gallegos, M., Seron, C., Micheloud, D., Gomez, J., Tenorio-Abreu, A., Ramos, M., Molina, M., Huidobro, S., Sanchez, E., Gordon, M., Fernandez, V., del Castillo, A., Marcos, M., Villanueva, B., Lopez, C., Rodriguez-Dominguez, M., Galan, J., Canton, R., Lietor, A., Rojo, S., Eiros, J., Hinojosa, C., Gonzalez, I., Torner, N., Banner, D., Leon, A., Cuesta, P., Rowe, T., & Kelvin, D. (2009). Th1 and Th17 hypercytokinemia as early host response signature in severe pandemic influenza Critical Care, 13 (6) DOI: 10.1186/cc8208 ; free full text (OA) at http://ccforum.com/content/pdf/cc8208.pdf
  2. Wynn, T. (2005). TH-17: a giant step from TH1 and TH2 Nature Immunology, 6 (11), 1069-1070 DOI: 10.1038/ni1105-1069
  3. Park, H., Li, Z., Yang, X., Chang, S., Nurieva, R., Wang, Y., Wang, Y., Hood, L., Zhu, Z., Tian, Q., & Dong, C. (2005). A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17 Nature Immunology, 6 (11), 1133-1141 DOI: 10.1038/ni1261
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NOT ONE RCT on Swine Flu or H1N1?! – Outrageous!

16 12 2009

Last week doctorblogs (Annabel Bentley) tweeted: “Outrageous- there isn’t ONE randomised trial on swine flu or #H1N1

Annabel referred to an article at Trust the Evidence, the excellent blog of the Centre for Evidence-Based Medicine (CEBM) in Oxford, UK.

In the article “Is swine flu the most over-published and over-hyped disease ever?Carl Heneghan first showed the results of a quick PubMed search using the terms ‘swine flu’ and ‘H1N1’: this yielded 4,475 articles on the subject, with approximately one third (1,437 articles) published in the last 7 months (search: November 27th). Of these 107, largely news articles, were published in the BMJ, followed by the Lancet and NEJM at 35 each.

Top News stories on H1N1 generated appr. 2000 to 4000 news articles each (in Google). Items included outbreak of a new form of ‘swine flu’ which prompted the United States and the World Health Organization to declare a public health emergency (April), Southern Hemisphere being mostly spared in the swine flu epidemic (May), Tamiflu, i.e. the effects of Tamiflu in children in the BMJ (co-authored by Carl) in August and the availability of the vaccine H1N1 vaccine clinics to offer seasonal flu shots in November.

According to Heneghan this must be the most over-hyped disease ever, and he wonders: “are there any other infections out there?”

Finally he ends with: Do you know what the killer fact is in all of this? There isn’t one randomized trial out there on swine flu or H1N1 – outrageous.”

My first thoughts were: “is H1N1 really so over-published compared to other (infectious) diseases?”, “Is it really surprising that there are no RCTs yet? The H1N1-pandemics just started a few months ago!” and even “are RCT’s really the study designs we urgently need right now?”

Now the severity of the H1N1 flu seems less than feared, it is easy to be wise. Isn’t is logic that there are a lot of “exploratory studies” first: characterization of the virus, establishing the spread of H1N1 around the world, establishing mortality and morbidity, and patterns of vulnerability among the population? It is also understandable that a lot of news articles are published, in the BMJ or in online newspapers. We want to be informed. In the Netherlands we now have a small outbreak of Q-fever, partly because the official approach was slow and underestimated the public health implications of Q-fever. So the public was really underinformed. That is worse than being “overexposed”.

News often spreads like wildfire, that is no news. When I google “US Preventive Services Task Force” (who issued the controversial US breast cancer screening guidelines last month) 2,364 hits still pop up in Google News (over the last month). All papers and other news sources echo the news. 2,000 hits are easily reached.

4,475 PubMed articles on ‘swine flu’ and ‘H1N1’ isn’t really that much. When I quickly search PubMed for the rather “new” disease Q-fever I get 3,752 hits, a search for HPV (Alphapapillomavirus OR papilloma infections OR HPV OR human papilloma virus) gives 19,543 hits (1,330 over the last 9 months), and a quick search for (aids) AND “last 9 months”[edat] yields 4,073 hits!

The number of hits alone doesn’t mean much, certainly not if news, editorials and comments are included. But lets go to the second comment, that there is “not ONE RCT on H1N1.”

Again, is it reasonable to expect ONE RCT published and included in PubMed over a 9 month period? Any serious study takes time from concept to initiation, patient-enrollment, sufficient follow-up, collection of data, writing and submitting the article, peer review, publication, inclusion in PubMed and assignment of MeSH-terms (including the publication type “Randomized Controlled Trial”).

Furthermore RCTs are not always the most feasible or appropriate study designs for answering certain questions. For instance for questions related to harm, etiology, epidemiology, spreading of virus, characteristics, diagnosis and prognosis. RCTs may be most suitable to evaluate the efficacy of treatment or prevention interventions. Thus in case of H1N1 the efficacy of vaccines and of neuraminidase inhibitors to prevent or treat H1N1 flu. However, it may not always be ethical to do so (see below).

I’ve repeated the search, and using prefab “My NCBI filters” for RCTs discussed before I get the following results:

Using the Randomized Controlled Trials limits in PubMed I do get 7 hits, and using broader filters, like the Therapy/Narrow Filter under  Clinical Queries I even find 2 more RCTs that have not yet been indexed by PubMed. With the Cochrane Highly sensitive Filter even more hits are obtained, most of which are “noise”, inherent to the use of a broad filter.

The found RCTs are safety/immunogenicity/stability studies of subunit or split vaccines to H1N1, H3N2, and B influenza strains. This means they are not restricted to H1N1, but this is true for the entire set of H1N1 publications. 40 of the 1443 hits are even animal studies. Thus the total number of articles dealing with H1N1 only -and in humans- is far less than 1443.
By the way, one of the 15 H1N1-hits in PubMed obtained with the SR-filter (see Fig) is a meta-analysis of RCTs in the BMJ, co-authored by Heneghan. It is not about H1N1, but contains the sentence: “Their (neuraminidase inhibitors) effects on the incidence of serious complications, and on the current A/H1N1 influenza strain remain to be determined.”

More important, if studies have been undertaken in this field they are probably not yet published. Thus, the place to look is a clinical trials register, like Clinical trials.gov (http://clinicaltrials.gov/), The International Clinical Registry Platform Search Portal at the WHO (www.who.int/trialsearch) , national or pharmaceutical industry trials registers.

A search for H1N1 OR swine flu in Clinical trials.gov, that offers the best searching functions, yields 132 studies, of which 116 were first recieved this year.

Again, most trials concern the safety and efficacy of H1N1 vaccines and include the testing of vaccines on subgroups, like pregnant women, children with asthma and people with AIDS. 30 trials are phase III.
Narrowing the search to H1N1
OR swine flu | neuraminidase inhibitors OR oseltamivir OR zanamivir (treatment filled in in the filed “Interventions”) yields 8 studies. One of the studies is a phase III trial.

This yield doesn’t seem bad per se. However, numbers of trials don’t mean a lot and a more pertinent issue is, whether the most important and urgent questions are investigated.

Three issues are important with respect to interventions:

  1. Are H1N1 vaccines safe and immunogenic? in subpopulations?
  2. Do H1N1 vaccines lower morbidity and mortality due to the H1N1 flu?
  3. Are neuraminidase inhibitors effective in preventing or treating H1N1 flu?
Question [1] will be answered by current trials.
Older Cochrane Reviews on the seasonal influenza flu (and updates) cast doubt on the efficacy of [2] vaccines (see the [poor*] Atlantic news article) ànd [2] neuraminidase inhibitors in children (Cochrane 2007 and BMJ 2009) ànd adults  (Cochrane 2006, update 2008 and BMJ 2009) against symptoms or complications of the seasonal flu. The possibility has even been raised that seasonal flu shots are linked to swine flu risk.
However, the current H1N1 isn’t a seasonal flu. It is a sudden, new pandemic that requires different actions. Overall H1N1 isn’t as deadly as the regular influenza strains, but it hits certain people harder: very young kids, people with asthma and pregnant women. About the latter group, Amy Tuteur (obstetrician-gynecologist blogging at The Skeptical OB) wrote a guest post at Kevin MD:
(…) the H1N1 influenza has had an unexpectedly devastating impact among pregnant women. According to the CDC, there have been approximately 700 reported cases of H1N1 in pregnant women since April.** Of these, 100 women have required admission to an intensive care unit and 28 have died. In other words, 1 out of every 25 pregnant women who contracted H1N1 died of it. By any standard, that is an appalling death rate. (……)
To put it in perspective, the chance of a pregnant woman dying from H1N1 is greater than the chance of a heart patient dying during triple bypass surgery. That is not a trivial risk.
The H1N1 flu has taken an extraordinary toll among pregnant women. A new vaccine is now available. Because of the nature of the emergency, there has not been time to do any long term studies of the vaccine. Yet pregnant women will need to make a decision as soon as possible on whether to be vaccinated. (Emphasis mine)
…. Given the dramatic threat and the fact that we know of no unusual complications of vaccination, the decision seems clear. Every pregnant woman should get vaccinated as soon as possible.
Thus the anticipated risks must be balanced against the anticipated benefits, Amy urges pregnant women to get vaccinated, even though no one can be sure about side effects ànd about the true efficacy of the vaccine.
For scientific purposes it would be best to perform a double randomized trial with half of a series of pregnant women receiving the vaccine, and the other half a placebo. This would provide the most rigid evidence for the true efficacy and safety of the vaccine.
However it would not be ethical to do so. As “Orac” of Orac Knows explains so well  in his post “Vaccination for H1N1 “swine” flu: Do The Atlantic, Shannon Brownlee, and Jeanne Lenzer matter?” RCTs are only acceptable from an ethical standpoint if we truly do not know whether one treatment is superior to another or a treatment is better than a placebo. There is sufficient reason to believe that vaccination for H1N1 will be more efficacious than “doing nothing”. Leaving a control group unvaccinated will certainly mean that a substantial percentage of pregnant women is going to die. To study the efficacy of the H1N1 among pregnant women observational studies (like cohort studies) are also suitable and more appropriate.
Among the studies found in ClinicalTrials.gov there are a few H1N1 Vaccine Clinical Studies in Pregnant Women, including RCTs. But these RCT’s never compare vaccinated women with a non-vaccinated women. All pregnant women are vaccinated, but the conditions vary.
In one Danish study the arms (study groups) are as follows:
Thus two doses of H1N1 with adjuvant are compared with a higher dose H1N1 without adjuvant. As a control non-pregnant women are vaccinated with the adjuvant H1N1.*** The RCT is performed within a prospective, birth-cohort study recruiting 800 pregnant mothers between Q1- 2009 and Q4-2010. As a natural control women pregnant in the H1N1 season (Q4) will be compared with women outside the season. Please note that the completion date of this study will be 2012, thus we will have to wait a number of years before the study describing the results will be found in PubMed….
To give an impression of the idea behind the study, here is the summary of that trial in the register (not because it is particularly outstanding, but to highlight the underlying thoughts):
“Pregnant women are at particular risk during the imminent H1N1v influenza pandemic. The new H1N1v virus requires urgent political and medical decisions on vaccination strategies in order to minimize severe disease and death from this pandemic. However, there is a lack of evidence to build such decisions upon. A vaccine will be provided in the fourth quarter of 2009, but there is little knowledge on the immunogenicity. Particularly its clinical effectiveness and duration of immunity in pregnant women and their newborn infants is unknown. Therefore, it will be important to study the optimal vaccination regimens with respect to dosing and use of adjuvant to decide future health policies on vaccination of pregnant women. We have a unique possibility to study these aspects of H1N1v infection in pregnant women in our ongoing unselected, prospective, birth-cohort study recruiting 800 pregnant mothers between Q1- 2009 and Q4-2010. Pregnant women from East-Denmark are being enrolled during the 2nd trimester and their infant will undergo a close clinical follow-up. The H1N1v pandemic is expected to reach Denmark Q4-2009. The timing of this enrollment and the imminent pandemic allows for an “experiment of nature” whereby the first half of the mothers completes pregnancy before the H1N1v pandemic. The other half of this cohort will be pregnant while H1N1v is prevalent in the community and will require H1N1v vaccination.The aim of this randomized, controlled, trial is to compare and evaluate the dose-related immune protection conferred by vaccine and adjuvant (Novartis vaccine Focetria) in pregnant women and non-pregnant women. In addition the protocol will assess the passive immunity conferred to the newborn from these vaccine regimes. The study will provide evidence-based guidance for health policies on vaccination for the population of pregnant women during future H1N1v pandemics.”
Although with regard to H1N1-vaccination, appropriate studies are being done, it is feasible that certain measures might not be appropriate on basis of what we know. For instance, pretreating people in the non-risk groups (healthy young adults) with neuraminidase-inhibitors, because they are “indispensable employees”. Perhaps Heneghan, who as you remember is a co-author of the BMJ paper on neuraminidase -inhibitors in children with the seasonal flu, was thinking of this when writing his post.
If Heneghan would have directed his arrows at certain interventions in certain circumstances in certain people he might have had a good point, but now his arrows don’t hit any target. Revere from Effect Measure and Orac from Orac Knows might well have diagnosed him as someone who suffers from “methodolatry,” which is, as Revere puts it, the “profane worship of the randomized clinical trial as the only valid method of investigation.”
* But see the excellent post of Orac who trashes the Atlantic paper in Flu vaccination: Do The Atlantic, Shannon Brownlee, and Jeanne Lenzer matter? (scienceblogs.com). He also critiques the attitude of the Cochrane author Jefferson, who has a different voice in the media compared to the Cochrane Reviews he co-authors. Here he is far more neutral.
** There is no direct link to the data in the post. I’m not sure whether all pregnant women in the US are routinely tested for H1N1. (if not the percentage of H1N1 deaths among H1N1 infected pregnant women might be overestimated)
***In the US, vaccins given to pregnant women are without adjuvant.


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