Penicillium marneffei infection and impaired IFN-γ immunity in humans with autosomal-dominant gain-of-phosphorylation STAT1 mutations
2013; Elsevier BV; Volume: 133; Issue: 3 Linguagem: Inglês
10.1016/j.jaci.2013.08.051
ISSN1097-6825
AutoresPamela Lee, Huawei Mao, Wanling Yang, Koon-Wing Chan, M. Ho, Tsz-Leung Lee, Jasper Fuk‐Woo Chan, Patrick C. Y. Woo, Wenwei Tu, YL Lau,
Tópico(s)Antifungal resistance and susceptibility
ResumoPenicillium marneffei is a pathogenic fungus endemic in Southeast Asia. P marneffei was an extremely rare pathogen in humans before the HIV epidemic, but following the exponential rise in the prevalence of HIV in Southeast Asia, penicilliosis emerged as a clinically significant opportunistic infection and is classified as an AIDS-defining illness.1Supparatpinyo K. Khamwan C. Baosoung V. Nelson K.E. Sirisanthana T. Disseminated Penicillium marneffei infection in southeast Asia.Lancet. 1994; 344: 110-113Abstract PubMed Scopus (503) Google Scholar Less commonly, penicilliosis occurs in patients with other immunodeficiencies, such as severe combined immunodeficiency, common variable immunodeficiency, hyper-IgM syndrome, hyper-IgE syndrome, the presence of anti-IFN-γ autoantibody, diabetes mellitus, immunosuppressive therapy, and solid organ or hematopoietic stem cell transplant.1Supparatpinyo K. Khamwan C. Baosoung V. Nelson K.E. Sirisanthana T. Disseminated Penicillium marneffei infection in southeast Asia.Lancet. 1994; 344: 110-113Abstract PubMed Scopus (503) Google Scholar, 2Tang B.S. Chan J.F. Chen M. Tsang O.T. Mok M.Y. Lai R.W. et al.Disseminated penicilliosis, recurrent bacteremic nontyphoidal salmonellosis, and burkholderiosis associated with acquired immunodeficiency due to autoantibody against gamma interferon.Clin Vaccine Immunol. 2010; 17: 1132-1138Crossref PubMed Scopus (78) Google Scholar Affected individuals often have disseminated disease with rapid progression to multiorgan failure and death.We previously reported 5 Chinese HIV-negative children and teenagers with disseminated penicilliosis. Four had coexisting chronic mucocutaneous candidiasis (CMC) since infancy, and 1 of them was genetically confirmed to have autosomal-dominant (AD) hyper-IgE syndrome. For the remaining 3 patients, a search for genetic defects in CARD9, AIRE, STAT3, IL12B, IL12RB1, and IFNGR1 was unrevealing.3Lee P.P. Chan K.W. Lee T.L. Ho M.H. Chen X.Y. Li C.H. et al.Penicilliosis in children without HIV infection–are they immunodeficient?.Clin Infect Dis. 2012; 54: e8-19Crossref PubMed Scopus (52) Google Scholar The coexistence of CMC and systemic penicilliosis suggested a possible functional defect of TH17 immune response in these patients. Recently, AD gain-of-function missense mutations of STAT1 have been identified in several multiplex kindreds displaying CMC, autoimmunity, and squamous cell carcinoma.4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (484) Google Scholar, 5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar, 6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 7Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (81) Google Scholar, 8Toth B. Mehes L. Tasko S. Szalai Z. Tulassay Z. Cypowyj S. et al.Herpes in STAT1 gain-of-function mutation.Lancet. 2012; 379: 2500Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Soltész B. Tóth B. Shabashova N. Bondarenko A. Okada S. Cypowyj S. et al.New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.J Med Genet. 2013; 50: 567-578Crossref PubMed Scopus (79) Google Scholar We hypothesized STAT1 as a candidate gene, and we sought to determine the cellular response to STAT1 activation in these patients. Consent for genetic diagnosis and functional studies was obtained from parents, and the study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster.Patients 1, 2, and 3 were unrelated Chinese children, and their clinical presentations and immunologic profile were previously reported in detail.3Lee P.P. Chan K.W. Lee T.L. Ho M.H. Chen X.Y. Li C.H. et al.Penicilliosis in children without HIV infection–are they immunodeficient?.Clin Infect Dis. 2012; 54: e8-19Crossref PubMed Scopus (52) Google Scholar The core features and genetic findings of the patients and their parents are listed in Table I and Fig E1 (in the Online Repository at www.jacionline.org). Heterozygous missense mutation in STAT1 was identified by Sanger sequencing in patient 1 (c.800C>T, p.A267V) and patient 3 (c.863C>T, p.T288I) and total exome sequencing in patient 2 (c.1074G>T, p.L358F; see the Online Repository at www.jacionline.org). p.A267V is a known mutation while p.T288I and p.L358F are novel, but missense mutations involving the same amino acid residues (p.T288A and p.L358W) were reported in patients with CMC.4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (484) Google Scholar, 5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar, 6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar Multiple sequence alignment of STAT1 orthologs (HomoloGene, NCBI) showed that all residues are highly conserved in animals except zebrafish for A267 and T288 and chicken for L358.Table ICore clinical features of 3 patients with systemic Penicillium marneffei infection and STAT1 mutationsPatient 1Patient 2Patient 3SexMaleFemaleFemaleAge of presentationInfancyInfancyInfancyFamily historyNil of significanceNil of significanceNil of significanceInfections FungusCMC, disseminated P marneffeiCMC, Candida albicans and Candia tropicalis otitis externa, disseminated P marneffeiCMC, disseminated P marneffei, disseminated aspergillosis BacteriaNil documentedRecurrent sinopulmonary infectionsRecurrent sinopulmonary infections MycobacteriaNil documentedNil documentedMycobacterium tuberculosis lymphadenopathy VirusNil documentedH1N1 influenza A respiratory infection with prolonged carriage, CMV pneumonitisRecurrent herpes zoster reactivation, EBV-associated hemophagocytosisMutation Nucleotide changec.800C>Tc.1074G>Tc.863C>T Amino acid changep.A267Vp.L358Fp.T288I DomainCoiled-coil domainDNA-binding domainCoiled-coil domain Carrier status of parentsNot carrierNot carrierMother not carrier (father not checked) Open table in a new tab Missense mutations affecting the STAT1 coiled-coil domain identified in patients with AD-CMC have been demonstrated to be gain-of-function mutants with increased tyrosine-701 residue phosphorylation and enhanced γ-activated sequence promoter binding activity.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar We compared the level of STAT1 phosphorylation in patients with that in healthy controls by flow cytometric analysis of intracellular phosphorylated STAT1 (pSTAT1). PBMCs from patients and controls were stimulated with recombinant human IFN-α (40,000 IU/mL) or IFN-γ (5,000 IU/mL) for 20 minutes. Compared with normal controls, lymphocytes from all patients demonstrated a significantly higher percentage of pSTAT1+ cells and increased phosphorylation intensity in response to IFN-α and IFN-γ stimulation (Fig 1, A and B; see Fig E2 in the Online Repository at www.jacionline.org). The kinetics of STAT1 dephosphorylation was studied in patient 1. When treated with tyrosine kinase inhibitor, almost all STAT1 in control cells was dephosphorylated by 30 minutes whereas about 50% and 25% of STAT1 in patient cells remained phosphorylated at 30 and 60 minutes, respectively, indicating prolonged STAT1 phosphorylation in patient cells (Fig 1, C). A missense mutant affecting residue L358 was previously shown to cause delayed dephosphorylation as well.6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google ScholarNext, we determined the proportion of IFN-γ– and IL-17A–expressing T cells in PBMCs activated by overnight incubation with phorbol 12-myristate 13-acetate (100 ng/mL) and ionomycin (1 μg/mL) in the presence of Brefeldin A. Patients had significantly lower CD3+/IFN-γ+ T cells (14.8% ± 1.5% vs 43.3% ± 12.8%, P < .01) and CD3+/IL-17A+ T cells (0.30% ± 0.11% vs 2.15% ± 1.41%, P = .01; Fig 1, D) than did normal controls. Finally, we evaluated the capacity of IFN-γ production toward fungal stimulation in patient 1 and patient 2. PBMCs were cocultured with Candida albicans or P marneffei for 2 days, and supernatants were collected for IFN- γ assay (FlowCytomix; Bender MedSystems, Vienna, Austria). Compared with normal controls, patient 1 and patient 2 produced much lower IFN-γ toward both fungi (Fig 1, E). Production of other cytokines (IL-1β, IL-6, TNF-α, and MIP-1α) was studied in patient 1 and was found to be comparable with that in normal controls (see Fig E3 in this article's Online Repository at www.jacionline.org).Previous studies demonstrated that patients with CMC caused by gain-of-phosphorylation STAT1 mutations had impaired TH1 and TH17 response as a result of defective signaling through the IL-12 and IL-23 pathways.4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (484) Google Scholar, 5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar, 6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 7Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (81) Google Scholar, 10Smeekens S.P. Plantinga T.S. van de Veerdonk F.L. Heinhuis B. Hoischen A. Joosten L.A. et al.STAT1 hyperphosphorylation and defective IL12R/IL23R signaling underlie defective immunity in autosomal dominant chronic mucocutaneous candidiasis.PLoS One. 2011; 6: e29248Crossref PubMed Scopus (91) Google Scholar Majority of these gain-of-phosphorylation mutants are located in the coiled-coil domain and 2 in the DNA-binding domain.6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 7Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (81) Google Scholar Impaired dephosphorylation of STAT1 enhances gamma-interferon activation factor–dependent cellular response to IFN-α/β, IFN-γ, and IL-27, which are repressors of TH17 development from naive T cells. The enhanced response mediated by STAT1 probably impairs TH17 immunity.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google ScholarThe identification of STAT1 and STAT3 mutations in patients with systemic penicilliosis suggests the importance of TH1 and TH17 immune response against P marneffei. It is generally believed that P marneffei establishes diseases in the lungs following the inhalation of conidia, and disseminates in the form of intracellular yeast via the reticuloendothelial system. The activation of macrophages by IFN-γ is essential for their fungicidal activity against P marneffei through the production of nitric oxide. While P marneffei infection was self-limiting in wild-type mice, all IFN-γ knockout mice died of systemic mycosis.11Sisto F. Miluzio A. Leopardi O. Mirra M. Boelaert J.R. Taramelli D. Differential cytokine pattern in the spleens and livers of BALB/c mice infected with Penicillium marneffei: protective role of gamma interferon.Infect Immun. 2003; 71: 465-473Crossref PubMed Scopus (49) Google Scholar In humans, individuals with anti-IFN-γ autoantibody suffered from disseminated penicilliosis.2Tang B.S. Chan J.F. Chen M. Tsang O.T. Mok M.Y. Lai R.W. et al.Disseminated penicilliosis, recurrent bacteremic nontyphoidal salmonellosis, and burkholderiosis associated with acquired immunodeficiency due to autoantibody against gamma interferon.Clin Vaccine Immunol. 2010; 17: 1132-1138Crossref PubMed Scopus (78) Google Scholar Our experiments showed that lymphocytes of patient 1 and patient 2 exhibited defective IFN-γ production to P marneffei in vitro. To our knowledge, this study shows for the first time that a primary defect in IFN-γ and IL-17 immune response may be accountable for human P marneffei infection. Penicilliosis should be regarded as an indicator of underlying primary immunodeficiency in HIV-negative individuals after excluding secondary causes.It is worth noting that impaired IFN-γ and TH17 response in patients with gain-of-phosphorylation STAT1 mutations can predispose them to invasive mycosis as well as a range of bacterial and viral infections. Apart from penicilliosis, disseminated aspergillosis, candidemia, disseminated histoplasmosis, and recalcitrant cutaneous fusariosis were reported.6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 12Wang X. Lin Z. Gao L. Wang A. Wan Z. Chen W. et al.Exome sequencing reveals a signal transducer and activator of transcription 1 (STAT1) mutation in a child with recalcitrant cutaneous fusariosis.J Allergy Clin Immunol. 2013; 131: 1242-1243Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Patient 2 and patient 3 had recurrent sinopulmonary infections caused by respiratory viruses and encapsulated bacteria, which was also similarly described by Uzel et al6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar and Takezaki et al.7Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (81) Google Scholar Of note, patient 3 had tuberculous lymphadenitis, recurrent herpes zoster, and EBV-associated hemophagocytosis, supporting previous observation that AD gain-of-phosphorylation STAT1 mutations are associated with susceptibility to mycobacterial and herpes virus infections.8Toth B. Mehes L. Tasko S. Szalai Z. Tulassay Z. Cypowyj S. et al.Herpes in STAT1 gain-of-function mutation.Lancet. 2012; 379: 2500Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Autoimmunity such as hypothyroidism, autoimmune hepatitis, systemic lupus erythematosus, and type I diabetes mellitus, as well as malignancy such as esophageal carcinoma, can lead to significant morbidities to this group of patients. The infectious disease susceptibility and phenotypic spectrum of AD-CMC caused by STAT1 mutations are wider than previously believed, revealing the divergent roles of STAT1 in host-pathogen interaction, immune tolerance, and carcinogenesis. Penicillium marneffei is a pathogenic fungus endemic in Southeast Asia. P marneffei was an extremely rare pathogen in humans before the HIV epidemic, but following the exponential rise in the prevalence of HIV in Southeast Asia, penicilliosis emerged as a clinically significant opportunistic infection and is classified as an AIDS-defining illness.1Supparatpinyo K. Khamwan C. Baosoung V. Nelson K.E. Sirisanthana T. Disseminated Penicillium marneffei infection in southeast Asia.Lancet. 1994; 344: 110-113Abstract PubMed Scopus (503) Google Scholar Less commonly, penicilliosis occurs in patients with other immunodeficiencies, such as severe combined immunodeficiency, common variable immunodeficiency, hyper-IgM syndrome, hyper-IgE syndrome, the presence of anti-IFN-γ autoantibody, diabetes mellitus, immunosuppressive therapy, and solid organ or hematopoietic stem cell transplant.1Supparatpinyo K. Khamwan C. Baosoung V. Nelson K.E. Sirisanthana T. Disseminated Penicillium marneffei infection in southeast Asia.Lancet. 1994; 344: 110-113Abstract PubMed Scopus (503) Google Scholar, 2Tang B.S. Chan J.F. Chen M. Tsang O.T. Mok M.Y. Lai R.W. et al.Disseminated penicilliosis, recurrent bacteremic nontyphoidal salmonellosis, and burkholderiosis associated with acquired immunodeficiency due to autoantibody against gamma interferon.Clin Vaccine Immunol. 2010; 17: 1132-1138Crossref PubMed Scopus (78) Google Scholar Affected individuals often have disseminated disease with rapid progression to multiorgan failure and death. We previously reported 5 Chinese HIV-negative children and teenagers with disseminated penicilliosis. Four had coexisting chronic mucocutaneous candidiasis (CMC) since infancy, and 1 of them was genetically confirmed to have autosomal-dominant (AD) hyper-IgE syndrome. For the remaining 3 patients, a search for genetic defects in CARD9, AIRE, STAT3, IL12B, IL12RB1, and IFNGR1 was unrevealing.3Lee P.P. Chan K.W. Lee T.L. Ho M.H. Chen X.Y. Li C.H. et al.Penicilliosis in children without HIV infection–are they immunodeficient?.Clin Infect Dis. 2012; 54: e8-19Crossref PubMed Scopus (52) Google Scholar The coexistence of CMC and systemic penicilliosis suggested a possible functional defect of TH17 immune response in these patients. Recently, AD gain-of-function missense mutations of STAT1 have been identified in several multiplex kindreds displaying CMC, autoimmunity, and squamous cell carcinoma.4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (484) Google Scholar, 5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar, 6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 7Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (81) Google Scholar, 8Toth B. Mehes L. Tasko S. Szalai Z. Tulassay Z. Cypowyj S. et al.Herpes in STAT1 gain-of-function mutation.Lancet. 2012; 379: 2500Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Soltész B. Tóth B. Shabashova N. Bondarenko A. Okada S. Cypowyj S. et al.New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe.J Med Genet. 2013; 50: 567-578Crossref PubMed Scopus (79) Google Scholar We hypothesized STAT1 as a candidate gene, and we sought to determine the cellular response to STAT1 activation in these patients. Consent for genetic diagnosis and functional studies was obtained from parents, and the study was approved by the Institutional Review Board of The University of Hong Kong/Hospital Authority Hong Kong West Cluster. Patients 1, 2, and 3 were unrelated Chinese children, and their clinical presentations and immunologic profile were previously reported in detail.3Lee P.P. Chan K.W. Lee T.L. Ho M.H. Chen X.Y. Li C.H. et al.Penicilliosis in children without HIV infection–are they immunodeficient?.Clin Infect Dis. 2012; 54: e8-19Crossref PubMed Scopus (52) Google Scholar The core features and genetic findings of the patients and their parents are listed in Table I and Fig E1 (in the Online Repository at www.jacionline.org). Heterozygous missense mutation in STAT1 was identified by Sanger sequencing in patient 1 (c.800C>T, p.A267V) and patient 3 (c.863C>T, p.T288I) and total exome sequencing in patient 2 (c.1074G>T, p.L358F; see the Online Repository at www.jacionline.org). p.A267V is a known mutation while p.T288I and p.L358F are novel, but missense mutations involving the same amino acid residues (p.T288A and p.L358W) were reported in patients with CMC.4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (484) Google Scholar, 5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar, 6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar Multiple sequence alignment of STAT1 orthologs (HomoloGene, NCBI) showed that all residues are highly conserved in animals except zebrafish for A267 and T288 and chicken for L358. Missense mutations affecting the STAT1 coiled-coil domain identified in patients with AD-CMC have been demonstrated to be gain-of-function mutants with increased tyrosine-701 residue phosphorylation and enhanced γ-activated sequence promoter binding activity.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar We compared the level of STAT1 phosphorylation in patients with that in healthy controls by flow cytometric analysis of intracellular phosphorylated STAT1 (pSTAT1). PBMCs from patients and controls were stimulated with recombinant human IFN-α (40,000 IU/mL) or IFN-γ (5,000 IU/mL) for 20 minutes. Compared with normal controls, lymphocytes from all patients demonstrated a significantly higher percentage of pSTAT1+ cells and increased phosphorylation intensity in response to IFN-α and IFN-γ stimulation (Fig 1, A and B; see Fig E2 in the Online Repository at www.jacionline.org). The kinetics of STAT1 dephosphorylation was studied in patient 1. When treated with tyrosine kinase inhibitor, almost all STAT1 in control cells was dephosphorylated by 30 minutes whereas about 50% and 25% of STAT1 in patient cells remained phosphorylated at 30 and 60 minutes, respectively, indicating prolonged STAT1 phosphorylation in patient cells (Fig 1, C). A missense mutant affecting residue L358 was previously shown to cause delayed dephosphorylation as well.6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar Next, we determined the proportion of IFN-γ– and IL-17A–expressing T cells in PBMCs activated by overnight incubation with phorbol 12-myristate 13-acetate (100 ng/mL) and ionomycin (1 μg/mL) in the presence of Brefeldin A. Patients had significantly lower CD3+/IFN-γ+ T cells (14.8% ± 1.5% vs 43.3% ± 12.8%, P < .01) and CD3+/IL-17A+ T cells (0.30% ± 0.11% vs 2.15% ± 1.41%, P = .01; Fig 1, D) than did normal controls. Finally, we evaluated the capacity of IFN-γ production toward fungal stimulation in patient 1 and patient 2. PBMCs were cocultured with Candida albicans or P marneffei for 2 days, and supernatants were collected for IFN- γ assay (FlowCytomix; Bender MedSystems, Vienna, Austria). Compared with normal controls, patient 1 and patient 2 produced much lower IFN-γ toward both fungi (Fig 1, E). Production of other cytokines (IL-1β, IL-6, TNF-α, and MIP-1α) was studied in patient 1 and was found to be comparable with that in normal controls (see Fig E3 in this article's Online Repository at www.jacionline.org). Previous studies demonstrated that patients with CMC caused by gain-of-phosphorylation STAT1 mutations had impaired TH1 and TH17 response as a result of defective signaling through the IL-12 and IL-23 pathways.4van de Veerdonk F.L. Plantinga T.S. Hoischen A. Smeekens S.P. Joosten L.A. Gilissen C. et al.STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis.N Engl J Med. 2011; 365: 54-61Crossref PubMed Scopus (484) Google Scholar, 5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar, 6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 7Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (81) Google Scholar, 10Smeekens S.P. Plantinga T.S. van de Veerdonk F.L. Heinhuis B. Hoischen A. Joosten L.A. et al.STAT1 hyperphosphorylation and defective IL12R/IL23R signaling underlie defective immunity in autosomal dominant chronic mucocutaneous candidiasis.PLoS One. 2011; 6: e29248Crossref PubMed Scopus (91) Google Scholar Majority of these gain-of-phosphorylation mutants are located in the coiled-coil domain and 2 in the DNA-binding domain.6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 7Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (81) Google Scholar Impaired dephosphorylation of STAT1 enhances gamma-interferon activation factor–dependent cellular response to IFN-α/β, IFN-γ, and IL-27, which are repressors of TH17 development from naive T cells. The enhanced response mediated by STAT1 probably impairs TH17 immunity.5Liu L. Okada S. Kong X.F. Kreins A.Y. Cypowyj S. Abhyankar A. et al.Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis.J Exp Med. 2011; 208: 1635-1648Crossref PubMed Scopus (569) Google Scholar The identification of STAT1 and STAT3 mutations in patients with systemic penicilliosis suggests the importance of TH1 and TH17 immune response against P marneffei. It is generally believed that P marneffei establishes diseases in the lungs following the inhalation of conidia, and disseminates in the form of intracellular yeast via the reticuloendothelial system. The activation of macrophages by IFN-γ is essential for their fungicidal activity against P marneffei through the production of nitric oxide. While P marneffei infection was self-limiting in wild-type mice, all IFN-γ knockout mice died of systemic mycosis.11Sisto F. Miluzio A. Leopardi O. Mirra M. Boelaert J.R. Taramelli D. Differential cytokine pattern in the spleens and livers of BALB/c mice infected with Penicillium marneffei: protective role of gamma interferon.Infect Immun. 2003; 71: 465-473Crossref PubMed Scopus (49) Google Scholar In humans, individuals with anti-IFN-γ autoantibody suffered from disseminated penicilliosis.2Tang B.S. Chan J.F. Chen M. Tsang O.T. Mok M.Y. Lai R.W. et al.Disseminated penicilliosis, recurrent bacteremic nontyphoidal salmonellosis, and burkholderiosis associated with acquired immunodeficiency due to autoantibody against gamma interferon.Clin Vaccine Immunol. 2010; 17: 1132-1138Crossref PubMed Scopus (78) Google Scholar Our experiments showed that lymphocytes of patient 1 and patient 2 exhibited defective IFN-γ production to P marneffei in vitro. To our knowledge, this study shows for the first time that a primary defect in IFN-γ and IL-17 immune response may be accountable for human P marneffei infection. Penicilliosis should be regarded as an indicator of underlying primary immunodeficiency in HIV-negative individuals after excluding secondary causes. It is worth noting that impaired IFN-γ and TH17 response in patients with gain-of-phosphorylation STAT1 mutations can predispose them to invasive mycosis as well as a range of bacterial and viral infections. Apart from penicilliosis, disseminated aspergillosis, candidemia, disseminated histoplasmosis, and recalcitrant cutaneous fusariosis were reported.6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 12Wang X. Lin Z. Gao L. Wang A. Wan Z. Chen W. et al.Exome sequencing reveals a signal transducer and activator of transcription 1 (STAT1) mutation in a child with recalcitrant cutaneous fusariosis.J Allergy Clin Immunol. 2013; 131: 1242-1243Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar Patient 2 and patient 3 had recurrent sinopulmonary infections caused by respiratory viruses and encapsulated bacteria, which was also similarly described by Uzel et al6Uzel G. Sampaio E.P. Lawrence M.G. Hsu A.P. Hackett M. Dorsey M.J. et al.Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.J Allergy Clin Immunol. 2013; 131: 1611-1623Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar and Takezaki et al.7Takezaki S. Yamada M. Kato M. Park M.J. Maruyama K. Yamazaki Y. et al.Chronic mucocutaneous candidiasis caused by a gain-of-function mutation in the STAT1 DNA-binding domain.J Immunol. 2012; 189: 1521-1526Crossref PubMed Scopus (81) Google Scholar Of note, patient 3 had tuberculous lymphadenitis, recurrent herpes zoster, and EBV-associated hemophagocytosis, supporting previous observation that AD gain-of-phosphorylation STAT1 mutations are associated with susceptibility to mycobacterial and herpes virus infections.8Toth B. Mehes L. Tasko S. Szalai Z. Tulassay Z. Cypowyj S. et al.Herpes in STAT1 gain-of-function mutation.Lancet. 2012; 379: 2500Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Autoimmunity such as hypothyroidism, autoimmune hepatitis, systemic lupus erythematosus, and type I diabetes mellitus, as well as malignancy such as esophageal carcinoma, can lead to significant morbidities to this group of patients. The infectious disease susceptibility and phenotypic spectrum of AD-CMC caused by STAT1 mutations are wider than previously believed, revealing the divergent roles of STAT1 in host-pathogen interaction, immune tolerance, and carcinogenesis. Exome sequencing proceduresThree microgram of genomic DNA extracted from PBMCs from patient 2 was used for exon capture by using Agilent SureSelect Human All Exon 50Mb and library preparation according to standard procedures. A total of 157.5 million sequencing reads of 90bp in length were generated by Illumina HiSeq. The raw reads were mapped to human reference genome hg19 by using BWA (http://bio-bwa.sourceforge.net/). About 125 million reads were successfully mapped to human genome reference, of which 90.6 million reads were mapped to targeted regions, reaching a mean depth of 132 in the targeted regions. A total of 94.35% of the targeted regions were covered by at least 10-fold and 89.95% of the targeted regions were covered by at least 20-fold.Single nucleotide variants (SNVs) were called by using GATK (http://www.broadinstitute.org/gatk/), and indel were identified by using programs such as GATKindel, Pindel (https://trac.nbic.nl/pindel/), and Dindel (http://www.sanger.ac.uk/resources/software/dindel/). A total of 23,916 SNVs and 1,753 indels were identified for this individual.SNV calls with a quality phred score of more than 30, mapping quality phred score of more than 10, and coverage depth of more than 10 were kept for further analysis. An overall concordance between the exome SNV calls and Illumina SNP chip genotype calls was 0.997. Ti/Tv ratio for all the SNV calls reached 2.93, which is the norm for whole exome sequencing data.Of the SNV calls, their population frequency in dbSNP, 1000 Genome Project, NHLBI Exome Sequencing Project (ESP) ESP6500 data set (http://evs.gs.washington.edu/EVS/), and an internal database on exome sequencing was examined and relatively common variants considered unlikely to be related to the disease phenotype were excluded from further consideration. Variants that were considered high (frame shift mutations, splicing site mutations, start/stop gains and losses) and moderate functional impact (missense substitutions and nonframe shift insertion deletions) were further considered. Potential functional impact of the missense mutations was also evaluated by algorithms including SIFT, PolyPhen2, LJB_PolyP, LJB_MutationTaster, LJB_LRT, and those considered to be unlikely to have a strong functional impact on the protein structure and function were also removed from further consideration. After these procedures, 358 SNVs remained. Because of high false-positive calls on indels, only those called by all 3 algorithms (n = 49) were further evaluated. Considering these SNVs and indels together, 41 genes either have a homozygous variant or have 2 or more variants in the same gene, having a potential to be compound heterozygous mutations. But none of them seem to be particularly interesting based on their functional annotation, expression profile, and so forth.Fig E2STAT1 phosphorylation induced by IFN-α and IFN-γ. PBMCs of patients and controls were stimulated with IFN-α or IFN-γ for 20 minutes. The percentage of intracellular phosphorylated STAT1 (pSTAT1) expression in lymphocyte was analyzed by using flow cytometry. Representative histograms are presented, and the numbers in the right upper corner indicate the mean fluorescence intensity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Cytokine expression induced by Candia albicans and Penicillium marneffei (PM) stimulation. Individual PBMCs were cocultured with C albicans (multiplicity of infection [MOI] of 5) or P marneffei conidia (MOI of 1) for 48 hours. The cytokine production in the supernatant was examined by FlowCytomix according to manufacturer's instruction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Alignment of sequencing reads around the mutation in STAT1. This was performed by Integrative Genomics Viewer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Three microgram of genomic DNA extracted from PBMCs from patient 2 was used for exon capture by using Agilent SureSelect Human All Exon 50Mb and library preparation according to standard procedures. A total of 157.5 million sequencing reads of 90bp in length were generated by Illumina HiSeq. The raw reads were mapped to human reference genome hg19 by using BWA (http://bio-bwa.sourceforge.net/). About 125 million reads were successfully mapped to human genome reference, of which 90.6 million reads were mapped to targeted regions, reaching a mean depth of 132 in the targeted regions. A total of 94.35% of the targeted regions were covered by at least 10-fold and 89.95% of the targeted regions were covered by at least 20-fold. Single nucleotide variants (SNVs) were called by using GATK (http://www.broadinstitute.org/gatk/), and indel were identified by using programs such as GATKindel, Pindel (https://trac.nbic.nl/pindel/), and Dindel (http://www.sanger.ac.uk/resources/software/dindel/). A total of 23,916 SNVs and 1,753 indels were identified for this individual. SNV calls with a quality phred score of more than 30, mapping quality phred score of more than 10, and coverage depth of more than 10 were kept for further analysis. An overall concordance between the exome SNV calls and Illumina SNP chip genotype calls was 0.997. Ti/Tv ratio for all the SNV calls reached 2.93, which is the norm for whole exome sequencing data. Of the SNV calls, their population frequency in dbSNP, 1000 Genome Project, NHLBI Exome Sequencing Project (ESP) ESP6500 data set (http://evs.gs.washington.edu/EVS/), and an internal database on exome sequencing was examined and relatively common variants considered unlikely to be related to the disease phenotype were excluded from further consideration. Variants that were considered high (frame shift mutations, splicing site mutations, start/stop gains and losses) and moderate functional impact (missense substitutions and nonframe shift insertion deletions) were further considered. Potential functional impact of the missense mutations was also evaluated by algorithms including SIFT, PolyPhen2, LJB_PolyP, LJB_MutationTaster, LJB_LRT, and those considered to be unlikely to have a strong functional impact on the protein structure and function were also removed from further consideration. After these procedures, 358 SNVs remained. Because of high false-positive calls on indels, only those called by all 3 algorithms (n = 49) were further evaluated. Considering these SNVs and indels together, 41 genes either have a homozygous variant or have 2 or more variants in the same gene, having a potential to be compound heterozygous mutations. But none of them seem to be particularly interesting based on their functional annotation, expression profile, and so forth.
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