Tuberculosis and impaired IL-23–dependent IFN-γ immunity in humans homozygous for a common TYK2 missense variant
Summary (3 min read)
INTRODUCTION
- About a quarter of the world’s population is infected with Mycobacterium tuberculosis, but this bacterium causes tuberculosis in less than 10% of infected individuals, generally within 2 years of infection (a situation referred to here as primary tuberculosis) (1–3).
- These results suggest that homozygosity for P1104A, which is still present in about 1/600 Europeans and between 1/10,000 and 1/1000 individuals in other regions of the world, with the exception of East Asia, where the allele is almost absent, has been a major human genetic determinant of primary tuberculosis during the course of human history.
- Like patients with complete TYK2 and IL-12R1 deficiencies, P1104A homozygotes did not respond to IL-23, in terms of IFN- production, as shown by comparison with healthy controls.
Whole blood ***
- TYK2 P1104A patients thus displayed impaired IL-23–mediated IFN- immunity.
- The authors then studied the capacity of naïve CD4+.
- These cells were unable to produce IL-17A/ IL-17F, consistent with the impairment of IL-23 signaling, as observed in IL-12R1– and TYK2–deficient patients (Fig. 6D).
- T cells from P1104A homozygotes were able to differentiate into IFN-–producing TH1 cells in an IL-12– dependent manner (TAE beads and IL-12), like control cells and cells from IL-23R–deficient patients, but unlike cells from TYK2-, IL-12R1–, and IL-12R2–deficient patients (Fig. 6D and companion paper).
DISCUSSION
- In conclusion, homozygosity for TYK2 P1104A confers a predisposition to severe mycobacterial diseases, including MSMD and, more frequently, primary tuberculosis.
- Several genome-wide association studies have shown that homozygosity for TYK2 P1104A has a strong protective effect (ORs ranging from 0.1 to 0.3) against various autoinflammatory or autoimmune conditions (41).
- The gradual decline of the P1104A allele in the European continent, which requires further investigation using more ancient DNA samples from different geographic locations and epochs, suggests that tuberculosis has been continuously endemic from the Neolithic until the middle of the 20th century (56).
- Genetic testing before travels into endemic areas may, however, be warranted.
- This observation has important clinical implications, because injections of recombinant IFN- would probably be beneficial in these patients, as it is in patients with IL-12R1 deficiency (12, 19, 88).
MATERIALS AND METHODS Study design
- The authors studied the contributions of two common TYK2 missense variants, I684S and P1104A, to predisposition to mycobacterial diseases.
- The authors screened their WES database including 463 patients with MSMD, 454 with tuberculosis, and 2835 with non-mycobacterial infections used as controls, as well as the WES data of the 2504 from the 1000 Genomes Project (a total of 5339 controls).
- The authors tested the association of the two TYK2 variants with MSMD and tuberculosis using a logistic regression model including the first three principal components of the PCA to account for the ethnic heterogeneity of the cohorts.
- The authors analyzed the occurrence of negative selection acting on the two TYK2 variants by testing whether their frequency in Europeans has decreased more than other variants that were in the same frequency range 4000 years ago.
- The authors also tested the patient’s primary leukocytes.
Ethics statement
- This study was conducted in accordance with the Helsinki Declaration, with written informed consent obtained from the patients’ families.
- Approval for this study was obtained from the French Ethics Committee “Comité de Protection des Personnes,” The French National Agency for Medicine and Health Product Safety (ANSM) and the Institut National de la Santé et de la Recherche Médicale in France, and the Rockefeller University Institutional Review Board (IRB), New York, USA.
Whole-blood activation experiments
- Venous blood samples from controls and patients were collected into heparin-containing tubes and processed according to a modified version of the protocol described by Feinberg et al. (89).
- ELISA was then performed on the collected supernatants, with the human IFN- ELISA Kit (Ready-SET-Go! from eBioscience or PeliPair from Sanquin), in accordance with the manufacturer’s instructions.
- The plasmids were used to transfect Phoenix-A packing cells to generate retroviral particles carrying each allelic variant.
- Positively transfected cells were selected with puromycin at a concentration of 2 g/ml until all the cells were positive for the surface expression of NGFR, as assessed by fluorescence-activated cell sorting with Alexa Fluor 647 anti-NGFR staining (BD Pharmingen).
Cell culture and stimulation
- EBV-B cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) .
- The cells were then starved for 2 hours by incubation in serum-free RPMI.
- A dose-response experiment was performed on EBV-B cells, with different concentrations of rhIL-23 ranging from Boisson-Dupuis et al., Sci. Immunol.
Western blotting
- Protein extracts were separated by SDS–polyacrylamide gel electrophoresis, and the resulting bands were electroblotted onto polyvinylidene difluoride membranes.
- Last, the blots were washed with washing buffer, and antibody binding was detected with the SuperSignal West Femto System (Thermo Fisher Scientific).
- S3D, immunoblots were revealed using enhanced chemiluminescence detection reagent (Western Lightning, PerkinElmer) and signals were acquired with Fuji ImageQuant LAS 4000.
- The level of phosphorylated band in control cells was set as 100.
- Statistical analysis was performed considering technical replicates for the transduced cells with the TYK2 alleles, and technical and biological replicates for EBV-B and HVS-T cell lines derived from controls and patients.
Population genetic analysis
- The authors investigated the occurrence of strong negative selection acting on the candidate TYK2 missense variants over the last few hundreds of generations, by comparing the current allele frequency of these variants in Europeans with that estimated from the low-coverage sequenced genomes of 22 individuals from Late Stone Age (LSA) Central Europe (52).
- Only variants that were still segregating in the current European population (CEU, TSI, or FIN from the 1000 Genomes Project) were considered in this analysis.
- The authors checked that these empirical observations were not biased due to sampling or sequencing errors, by performing 100,000 forward simulations under the Wright-Fisher neutral model (R code available upon request).
- The lower quantiles of the simulated and observed distributions were largely similar (Fig. 1E), suggesting that their analyses were unbiased.
WES and RNA-seq analyses
- WES was performed as previously described (47).
- Gene expression levels were estimated with TPM (transcripts per kilobase million).
- Gene expression profiles are expressed as the fold change in expression between the values obtained before and after stimulation.
Variant enrichment analysis
- The authors performed an enrichment analysis of TYK2 variants in their two cohorts of 463 MSMD patients and 454 tuberculosis patients.
- To account for the ethnic heterogeneity of the cohorts, the first three principal components of the PCA were systematically included in the logistic regression model, as previously described (48).
- Table S1. Summary of the TYK2 genotypes among the different cohorts of patients and healthy individuals.
- Raw data used to generate dot plots and bar graphs.
REFERENCES AND NOTES
- The global burden of latent tuberculosis infection: A re-estimation using mathematical modelling.
- Two rare disease-associated Tyk2 variants are catalytically impaired but signaling competent.
- The Laboratory of Human Genetics of Infectious Diseases was supported, in part, by grants from the French National Agency for Research (ANR) under the “Investissement d’avenir” program (grant no. ANR-10-IAHU-01), the TBPATHGEN project (grant no, also known as Funding.
- The Yale Center for Mendelian Genomics (UM1HG006504) is funded by the National Human Genome Research Institute.
Did you find this useful? Give us your feedback
Citations
1,659 citations
254 citations
139 citations
122 citations
121 citations
References
272,030 citations
30,684 citations
26,280 citations
15,744 citations
12,661 citations
Related Papers (5)
Frequently Asked Questions (11)
Q2. What is the reason for the impairment of IFN production in P1104A homozygo?
T cells of P1104A homozygotes have impaired IFN- production, due to their very weak response to IL-23, accounting for the susceptibility to MSMD or primary tuberculosis.
Q3. What is the role of TYK2 in antimycobacterial immunity?
the TYK2-dependent response to IL-23 that is disrupted by P1104A is essential for antimycobacterial IFN- immunity, but seems to be redundant for anti-fungal IL-17 immunity, given the absence of Candida infection in the patients described here.
Q4. What is the effect of TYK2 variants on cellular responses to IL-12?
The impact of TYK2 variants on cellular responses to IL-12 and IL-23 is irrelevant in nonhematopoietic cells, because the receptors for these cytokines are expressed only on leukocytes.
Q5. What is the CI for the TYK2 P1104A homozygos?
Several genome-wide association studies have shown that homozygosity for TYK2 P1104A has a strong protective effect (ORs ranging from 0.1 to 0.3) against various autoinflammatory or autoimmune conditions (41).
Q6. What grants were awarded to the Laboratory of Human Genetics of Infectious Diseases?
Funding: The Laboratory of Human Genetics of Infectious Diseases was supported, in part, by grants from the French National Agency for Research (ANR) under the “Investissement d’avenir” program (grant no. ANR-10-IAHU-01), the TBPATHGEN project (grant no.
Q7. What is the effect of negative selection on TYK2 P1104A?
The stronger selection operating on MEFV M694V, and to a lesser extent HFE C282Y, than on TYK2 P1104A is consistent with the inevitability of MF and hemochromatosis in patients with these mutations, whereas tuberculosis development also requires exposure to M. tuberculosis.
Q8. How did the authors determine the frequency of TYK2 variants in Europeans?
The authors analyzed the occurrence of negative selection acting on the two TYK2 variants by testing whether their frequency in Europeans has decreased more than other variants that were in the same frequency range 4000 years ago.
Q9. What is the common etiology of tuberculosis in Europe?
Between 1/10,000 and 1/1000 individuals are homozygous in endemic regions of the world (other than East Asia), where P1104A TYK2 is likely to define a strictly recessive but relatively common etiology of severe primary tuberculosis (about 0.5% of cases).
Q10. What was the phenotype of TYK2 in P1104A homozy?
In P1104A homozygous cells, the response to IFN- was modestly reduced in terms of JAK1, TYK2, STAT3, and STAT1 phosphorylation (Fig. 3C and fig.
Q11. What is the level of enrichment in homozygosity?
P1104A homozygosity was more enriched among patients with MSMD than among controls [P = 3.27 × 10−3; OR, 23.53; 95% confidence interval (CI), 2.9 to 483], and an even higher level of enrichment was observed among patients with tuberculosis (P = 8.37 × 10−8; OR, 89.31; 95% CI, 14.7 to 1725).