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

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.

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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]
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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.

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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.”

References

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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