Published online December 1, 2006
PEDIATRICS Vol. 118 No. 6 December 2006, pp. 2498-2503 (doi:10.1542/peds.2006-1845)

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ARTICLE

A Fast Procedure for the Detection of Defects in Toll-like Receptor Signaling

Horst von Bernuth, MD^a, Cheng-Lung Ku, MSc^a, Carlos Rodriguez-Gallego, MD^b, Shenying Zhang, MD^a, Ben-Zion Garty, MD^c, László Maródi, MD, PhD^d, Helen Chapel, MD^e, Maya Chrabieh^a, Richard L. Miller, PhD^f, Capucine Picard, MD, PhD^a,g, Anne Puel, PhD^a and Jean-Laurent Casanova, MD, PhD^a,h

^a Laboratory of Human Genetics of Infectious Diseases, University of Paris René Descartes, Institut National de la Santé et de la Recherche Médicale U550, Necker Medical School, Paris, France
^b Department of Immunology, Gran Canaria Dr Negrin Hospital, Las Palmas de Gran Canaria, Spain
^c Shneider Medical Center of Israel and Tel Aviv University Medical School, Tel Aviv, Israel
^d Department of Infectology and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
^e Department of Clinical Immunology, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford, United Kingdom
^f 3M Center, St Paul, Minnesota
^g Immunodeficiency Study Center
^h Pediatric Hematology-Immunology Unit, Necker Hospital, Paris, France

	ABSTRACT

TOP ABSTRACT PATIENTS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
OBJECTIVES. Inborn defects in Toll-like receptor signaling are recently described primary immunodeficiencies that predispose affected children to life-threatening infections. Patients with interleukin-1 receptor-associated kinase-4 deficiency are prone to invasive pneumococcal disease, and patients with UNC-93B deficiency are prone to herpes simplex virus encephalitis. These genetic disorders are underdiagnosed, partly because diagnosis currently requires expensive and time-consuming techniques available at only a few specialized centers worldwide. We, therefore, aimed to develop a cheap and fast test for the detection of defects in Toll-like receptor signaling.

PATIENTS AND METHODS. We used flow cytometry to evaluate the cleavage of membrane-bound L-selectin on granulocytes in 38 healthy controls and in 7 patients with genetically defined Toll-like receptor signaling defects (5 patients with interleukin-1 receptor-associated kinase-4 deficiency and 2 patients with UNC-93B deficiency), on activation with various Toll-like receptor agonists.

RESULTS. Impaired L-selectin shedding was observed with granulocytes from all of the interleukin-1 receptor-associated kinase-4-deficient patients on activation with agonists of Toll-like receptors 1/2, 2/6, 4, 7, and 8 and with granulocytes from all of the UNC-93B-deficient patients on activation with agonists of Toll-like receptors 7 and 8. All of the healthy controls responded to these stimuli.

CONCLUSIONS. The assessment of membrane-bound L-selectin cleavage on granulocytes by flow cytometry may prove useful for the detection of primary immunodeficiencies in the Toll-like receptor pathway, such as interleukin-1 receptor-associated kinase-4 deficiency and UNC-93B deficiency. This procedure is cheap and rapid. It may, therefore, be suitable for routine testing worldwide in children with invasive pneumococcal disease and in patients with herpes simplex encephalitis.

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Key Words: innate immunity • interleukin 1 receptor-associated kinase-4 deficiency • L-selectin • primary immunodeficiency • Toll-like receptors • UNC-93B deficiency

Abbreviations: TLR—Toll-like receptor • IRAK-4—interleukin-1-receptor-associated-kinase-4 • TNF-—tumor necrosis factor • IL—interleukin • LPS—lipopolysaccharide • ELISA—enzyme-linked immunosorbent assay • CD62L—L-selectin • PMA—phorbolmyristyl acetate • Pam₂CSK₄—synthetic diacylayed lipopeptide • Pam₃CSK₄—synthetic triacylayed lipopeptide • R-848—resiquimod • 3M-2—synthetic agonist of TLR8 • 3M-13—synthetic agonist of TLR7

Children with infectious diseases as diverse as invasive pneumococcal disease and herpes simplex encephalitis may suffer from novel primary immunodeficiencies in the early Toll-like receptor (TLR) signaling pathway.^1,2 Interleukin-1 receptor-associated kinase-4 (IRAK-4) deficiency has been diagnosed in 28 patients and confers a predisposition to invasive disease caused by pyogenic bacteria, notably Streptococcus pneumoniae and, to a lesser extent, Staphylococcus aureus (unpublished data).^1,3–11 UNC-93B deficiency seems to confer a selective predisposition to meningo-encephalitis caused by herpes simplex virus, but the range of pathogens to which patients are susceptible may actually be much larger, because only 2 patients have been identified to date.² The 10 human TLRs signal via IRAK-4, which is redundant for the TLR3- and TLR4-mediated induction of antiviral interferons.^8,12,13 IRAK-4 is crucial for the early induction of proinflammatory cytokines, such as tumor necrosis factor (TNF-), interleukin (IL)-1ß, and IL-6, by agonists from pyogenic bacteria via TLR1/2, TLR4, TLR5, TLR2/6, and TLR9.^14,15 Four TLRs (TLR3, TLR7, TLR8, and TLR9) require UNC-93B for signaling. These intracellular TLRs can be triggered by nucleic acids produced in the course of viral infection and are potent inducers of antiviral interferons.^2,16

IRAK-4 deficiency is associated with life-threatening, invasive, pyogenic bacterial diseases, typically caused by Gram-positive species, such as S pneumoniae and S aureus (C.-L.K. and J.-L.C., unpublished data, 2006),^1,3–11 and, more rarely, by Gram-negative species.^1,3,4,7,10 Infections invariably strike in infancy and early childhood, most frequently presenting as meningitis, arthritis, and/or septicemia.^1,3–11 Other typical features include mild clinical and biological inflammatory reactions despite life-threatening infections^1,3,6,17 and delayed separation of the umbilical cord.⁹ Since its initial description in 2003, 28 patients from 18 kindreds have been identified in 11 countries worldwide (C.-L.K. and J.-L.C., unpublished data, 2006).^1,3–11 Known patients with IRAK-4 deficiency display no particular susceptibility to mycobacterial, fungal, or viral infections. In contrast, UNC-93B deficiency is associated with herpes simplex virus encephalitis but not with other infectious diseases (bacterial infections, in particular).² The range of pathogens known to cause clinical disease may expand, however, because UNC-93B deficiency has only recently been discovered, with only 2 patients reported to date.

IRAK-4 deficiency and UNC-93B deficiency are both probably underdiagnosed. They have a narrower infection spectrum than conventional primary immunodeficiencies, often limited to invasive pyogenic disease in IRAK-4 deficiency and to herpes simplex encephalitis in UNC-93B deficiency. Moreover, no immunologic abnormality is detected in commonly available assays of myeloid and lymphoid cells. IRAK-4 deficiency is currently detected by measuring the in vitro production of TNF- or IL-6 by whole blood cells or peripheral blood mononuclear cells stimulated with TLR agonists (eg, lipopolysaccharide [LPS]), either in the supernatant by enzyme-linked immunosorbent assay (ELISA)^{1,4,7,8,10–11,18} or within cells by flow cytometry.⁹ Similarly, UNCB-93B deficiency is detected by assessing antiviral interferon production by peripheral blood mononuclear cells stimulated with TLR agonists.² These procedures are cumbersome, expensive, and slow, limiting the detection of IRAK-4 and UNC-93B deficiency to a small number of laboratories worldwide. We describe here a cheap and rapid method for the detection of defects in TLR signaling, based on the flow cytometry detection of L-selectin (CD62L) ectodomain-cleavage on the surface of granulocytes.

	PATIENTS AND METHODS

TOP ABSTRACT PATIENTS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Activation of Whole Blood Before Assessment of CD62L Expression
Heparin-treated blood was taken from 38 healthy control subjects and from 7 patients with genetically confirmed defects in TLR signaling (5 patients with IRAK-4 deficiency from France, England, Spain, Hungary, and Israel^{1,7,8,10–11} and 2 patients with UNC-93B deficiency from Portugal and France²) after informed consent had been obtained. The patients were from nonrelated kindreds. We incubated 100 µL of blood for 1 hour without stimulation or with TLR agonists, TNF-, or phorbolmyristyl acetate (PMA) at 37°C in a humidified atmosphere containing 5% CO₂. The following concentrations were used: synthetic diacylayed lipopeptide ([Pam₂CSK₄], agonist for TLR2/6, Invivogen, San Diego, CA, tlrl-pm2), 100 ng/mL; synthetic triacylayed lipopeptide ([Pam₃CSK₄], TLR1/2, Invivogen, tlrl-pms) 1 µg/mL; LPS (TLR4, Salmonella minnesota Re595 LPS, Sigma, St Louis, MO, L-9764), 100 ng/mL; flagellin (TLR5, Invivogen, tlrl-stfla), 1 µg/mL; synthetic agonist of TLR8 ([3M-2], TLR8, gift from 3M-Pharmaceuticals, St Paul, MN), 3 µg/mL; synthetic agonist of TLR7 ([3M-13] TLR7, gift from 3M-Pharmaceuticals), 30 µg/mL; resiquimod ([R-848], TLR7/TLR8, PharmaTech, Shanghai, China, specially provided), 3 µg/mL; TNF- (TNFR, R&D systems, Minneapolis, MN, 210-TA), 20 ng/mL; and PMA (Sigma, P-8139), 2 µg/mL. For all of the incubations except that with LPS, the cells were first incubated with 10 µg/mL of polymyxin B (Sigma, P-4932) for 20 minutes. We did not assess the response to polyinosine-polycytidylic acid, because TLR3 is not expressed on granulocytes.¹⁹ Neither did we analyze the response to unmethylated double-stranded CpG DNA, because optimal TLR9 expression on granulocytes requires previous treatment with granulocyte macrophage colony-stimulating factor.¹⁹ Finally, we did not assess the response to IL-1ß, because it has been shown that granulocytes do not modulate adhesion molecule expression in response to IL-1ß.²⁰

Analysis of CD62L Shedding
After the activation of whole blood, erythrocytes were lysed in a 1.3-M NH₄Cl, 100-mM KHCO₃, 1-mM ethylenediaminetetraacetic acid buffer, and cells were washed in Roswell Park Memorial Institute 1640. The contents of each tube were divided into 2 samples, 1 of which was incubated for 15 minutes on ice with the fluorescein isothiocyanate–conjugated anti-human CD62L antibody, the other being incubated with the respective isotype control (BD Biosciences Pharmingen, 555543 and 555748). The samples were then analyzed by flow cytometry, and the results were shown as histograms of events gated on granulocytes in an sideward scatter/forward scatter diagram, with the mean calculated in each case.

Activation of Whole Blood and Assessment of IL-6 Production
This assay was performed in 7 patients with known IRAK-4 deficiency as described previously in more detail.⁷ In brief, whole blood was diluted 1:1 with Roswell Park Memorial Institute 1640 without serum and incubated for 48 hours without any agonist, with LPS, final concentration 100 ng/mL, and PMA/Ionomycin (Sigma), final concentrations 10^–7 M/10^–5 M. IL-6 concentrations in the supernatant were assessed using a commercially available ELISA kit (Sanquin, NL, M9136).

	RESULTS

TOP ABSTRACT PATIENTS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
Granulocytes have been shown to display rapid cleavage of the ectodomain of membrane-bound CD62L on stimulation with the agonist of TLR4, LPS ("CD62L shedding").^19,21,22 We, therefore, investigated induced CD62L shedding (loss of CD62L expression) in 5 IRAK-4-deficient patients, in 2 patients with UNC-93B deficiency, and in 38 healthy control subjects, after whole-blood activation with LPS (an agonist of TLR4), Pam₃CSK₄ (TLR1/2), Pam₂CSK₄ (TLR2/6), flagellin (TLR5), 3M-13 (TLR7), R-848 (TLR7 and TLR8), 3M-2 (TLR8), TNF-, or PMA.

In all of the healthy control subjects, all of the IRAK-4-deficient patients, and all of the UNC-93B-deficient patients, the levels of CD62L expression on granulocytes after incubation with TNF- and PMA (induced CD62L shedding) were lower than the expression of CD62L on nonstimulated granulocytes (Figs 1 and 2). In contrast, the agonists Pam₃CSK₄ (TLR1/2), Pam₂CSK₄ (TLR2/6), LPS (TLR4), and R-848 (TLR7 and TLR8) induced strong CD62L shedding in all of the healthy control subjects tested but not in any of the IRAK-4-deficient patients (Figs 1 and 2). Furthermore, in both UNC-93B-deficient patients, the agonists Pam₃CSK₄ (TLR1/2), Pam₂CSK₄ (TLR2/6) and LPS (TLR4) induced strong CD62L shedding, whereas R-848 (TLR7 and TLR8) did not (Fig 2). CD62L shedding in response to flagellin (TLR5), 3M-13 (TLR7), and 3M-2 (TLR8) varied considerably among the healthy control subjects tested, making it impossible to draw conclusions from the comparison of control subjects and IRAK-4- and UNC-93B-deficient patients (data not shown).

View larger version (31K): [in this window] [in a new window]   FIGURE 1 Ectodomain cleavage of CD62L on granulocytes from an IRAK-4-deficient patient from Israel (right, IRAK4–/–) and a healthy control subject (left, control), on activation with agonists of TLR1/2 (PAM3CSK4), TLR2/6 (PAM2CSK4), TLR4 (LPS), TLR5 (flagellin), TLR7 (3M-13), TLR7 and TLR8 (R-848), TLR8 (3M-2), or TNF-. The black line shows the expression of CD62L on unstimulated granulocytes 48 hours after the taking of the blood sample and its transfer to the laboratory (spontaneous CD62L shedding). The red line shows the expression of CD62L after 1 hour of activation by different agonists (induced CD62L shedding). The CD62L signal was analyzed, with gating on granulocytes.

 

View larger version (10K): [in this window] [in a new window]   FIGURE 2 Summary of the results obtained for 38 healthy control subjects. Blood was taken <6 hours before the test in the Parisien area in 28 cases, and between 6 and 48 hours before testing (because of transfer of the sample to the laboratory from outside Paris), in 10 case subjects, 5 patients with IRAK-4 deficiency from France, England, Spain, Hungary, and Israel, and 2 patients with UNC-93B deficiency from Portugal and France. Each symbol represents the mean fluorescence intensity in fluorescine-1 of granulocytes stained with a fluorescein isothiocyanate–conjugated antibody directed against CD62L. Bars indicate the mean fluorescence intensity for the respective cohort. Granulocytes of 4/5 IRAK-4-deficient patients were tested >6 but <48 hours after the blood had been drawn. In 1 IRAK-4-deficient patient and in both UNC-93B-deficient patients, the test was performed <6 hours after the blood had been drawn.

 
The comparison of CD62L expression on nonstimulated granulocytes that were stained within a time shorter than 6 hours after the blood had been drawn (black circles) with nonstimulated granulocytes that had traveled 48 hours before having been stained (black squares) showed considerable levels of spontaneous CD62L shedding in some samples after travel (Fig 2). To validate our method further, we compared the CD62L shedding on granulocytes after stimulation with different agonists of TLRs for 1 hour to the production of IL-6 by whole blood after stimulation with LPS for 48 hours, the standard assay for the detection of IRAK-4 deficiency.^7,8,18 All of the IRAK-4-deficient patients showed both a severely impaired CD62L shedding on granulocytes and a profoundly impaired IL-6 production after activation by LPS (Figs 2 and 3).

View larger version (8K): [in this window] [in a new window]   FIGURE 3 IL-6 production by whole blood stimulated by LPS and PMA/Ionomycin for 48 hours in 8 healthy control subjects and in 9 patients with IRAK-4 deficiency.

 

	DISCUSSION

TOP ABSTRACT PATIENTS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
CD62L shedding on granulocytes stimulated with LPS was reported previously to be impaired in 1 patient with recurrent pyogenic infections²¹ 6 years before this patient was diagnosed as carrying a defect in TLR signaling (IRAK-4 deficiency).³ However, in this initial report, the response to only 1 TLR agonist (LPS) was tested, in only 1 IRAK-4-deficient patient, and with a single healthy control subject for comparison. It was, therefore, not possible to draw definitive conclusions from this observation as to whether the assessment of CD62L shedding on TLR activation can be used to detect various defects in TLR signaling.²¹ We, therefore, studied CD62L shedding on granulocytes stimulated by multiple TLRs in 38 healthy control subjects and in 7 patients with 2 different genetically confirmed defects in TLR signaling (5 IRAK-4-deficient patients and 2 patients with UNC-93B deficiency). In contrast to what was observed for all of the control subjects tested, no inducible CD62L shedding was detected on activation with Pam₃CSK₄ (TLR1/2), Pam₂CSK₄ (TLR2/6), LPS (TLR4), or R-848 (TLR7 and TLR8) in the 5 IRAK-4-deficient patients. Moreover, no inducible CD62L shedding was detected on activation with R-848 (TLR7 and TLR8) in the 2 patients with UNC-93B deficiency (Fig 2). Other TLR agonists (flagellin, 3M-13, and 3M-2) induced CD62L shedding in some but not all of the control subjects tested (data not shown) and were, therefore, of little diagnostic value.

Little is known about the pathways governing CD62L shedding.²² However, our results clearly indicate that CD62L shedding in response to TLR1/2, TLR2/6, and TLR4 is IRAK-4 dependent, whereas CD62L shedding in response to TLR7 and TLR8 depends on IRAK-4 and UNC-93B. Because IRAK-4 deficiency predisposes patients to pyogenic infections, and UNC-93B deficiency predisposes patients to herpes simplex encephalitis, we propose a method based on the assessment of granulocyte CD62L shedding after activation with 2 strong and easily available agonists of TLR4 and TLR7/TLR8, LPS and R-848, respectively, in all patients with pyogenic invasive infections and/or herpes simplex encephalitis. This assessment should be conducted as soon as possible after the taking of blood samples, because spontaneous CD62L autoshedding increases with time (Fig 2). Because induced CD62L shedding can easily be detected within 3 hours, this method is faster and cheaper than the intracellular assessment of cytokine production by flow cytometry⁹ or the even slower assessment of cytokine or interferon production by ELISA.^2,18 As shown by the comparison of LPS-induced CD62L shedding to the latter standard method of LPS activation of whole blood for 48 hours, both assays are equally reliable for the detection of defects in TLR signaling (Figs 2 and 3). However, the assessment of CD62L shedding requires only 500 µL of whole blood, making it particularly useful in pediatric immunology. In addition, impaired shedding of CD62L on TLR stimulation should also facilitate the identification of new, as yet unknown defects in early TLR signaling caused by mutations in genes other than IRAK4 and UNC93B (eg, MyD88 or IRAK1). We finally tested the granulocytes of 2 patients with known hypomorphic mutations in NEMO,²³ a gene that controls TLR signaling downstream of IRAK4. The whole blood of these patients was known to respond normally to activation by TLR agonists. Equally we observed no impaired CD62L shedding on activation by TLR agonists (data not shown). In contrast to patients with complete defects in TLR signaling, CD62L shedding may not prove useful for the detection of patients with hypomorphic mutations in NEMO.

	CONCLUSIONS

TOP ABSTRACT PATIENTS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 
The assessment of CD62L shedding on granulocytes after the stimulation of whole blood cells with the TLR4 agonist LPS and the TLR7/TLR8 agonist R-848 can be used for the rapid and cheap detection of immunodeficiencies caused by defects in TLR signaling, such as IRAK-4 deficiency and UNC-93B deficiency, but potentially also of other defects in early TLR signaling not yet characterized molecularly. Because testing for such immunodeficiencies is indicated in all infants and children with invasive pneumococcal and/or staphylococcal disease and in all patients with herpes simplex virus encephalitis, this method may serve as a useful tool for the pediatric immunologist.

	ACKNOWLEDGMENTS

 
Dr von Bernuth was supported by the Deutsche Forschungsgemeinschaft. Dr Puel was supported by the European Union. The Laboratory of Human Genetics of Infectious Diseases is supported in part by grants from 3M, Schlumberger, BNP Paribas, and the March of Dimes. Dr Casanova is an international scholar of the Howard Hughes Medical Institute.

We thank Laurent Abel, Lazaro Lorenzo, and all members of the Laboratory of Human Genetics of Infectious Diseases for helpful discussions. We especially thank Emanuelle Jouanguy for critically reading the article and for substantial suggestions. We are particularly grateful to the patients and their families for their participation in this study. We thank S. Ehl (University of Freiburg); A. Issekutz (Dalhouse University); D. Speert and S. Turvey (British Columbia Research Institute); C. Roifman (University of Toronto); C.K. Cunningham (Duke University); S. Holland (National Institutes of Health); and T. Hara (Kyushu University) for helpful discussions.

	FOOTNOTES

 
Accepted Aug 3, 2006.

Address correspondence to Horst von Bernuth, MD, or Cheng-Lung Ku, MSc, Laboratoire de Génétique Humaine des Maladies Infectieuses, Université de Paris René Descartes, Institut National de la Santé et de la Recherche Médicale U550, Faculté de Médecine Necker, 156 rue de Vaugirard, 75015 Paris, France. E-mail: vonbern{at}necker.fr or cheng-lung@necker.fr

The authors have indicated they have no financial relationships relevant to this article to disclose.

Dr von Bernuth and Mr Ku contributed equally to this study.

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TOP ABSTRACT PATIENTS AND METHODS RESULTS DISCUSSION CONCLUSIONS REFERENCES

 

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Web of Science (6) Citing Articles via Google Scholar Google Scholar Articles by von Bernuth, H. Articles by Casanova, J.-L. Search for Related Content PubMed PubMed Citation Articles by von Bernuth, H. Articles by Casanova, J.-L. Related Collections Genetics & Dysmorphology Related AAP Red Book topics: Herpes Simplex Social Bookmarking                         What's this?