Our patient is a 9-year-old girl, who was born as a second child to young healthy non-consanguineous parents, from Oaxaca, Mexico, without a family history of recurrent infections. She received the BCG vaccine during the first month of life, and at the age of 1 year, she developed an axillary adenitis at the site of the BCG vaccination and subsequent abscess, which was debrided with relief of symptoms. She was admitted to the hospital twice with symptoms of lower airways infection at the age of 1 and 3 years with no microorganism determination. During the second hospitalization, acid-fast bacilli (AFB) were reported in a sample of gastric juice; mycobacterial cultures were negative, a PPD test was also negative, and she did not receive any specific treatment. The patient presented also with recurrent episodes of oral candidiasis starting at the age of 8 months, and she received multiple drug treatments without achieving its complete eradication. At four and a half years old, the patient had infra-clavicular swelling with rubor and pain; she was diagnosed with a soft tissue abscess, and she was treated with Ceftriaxone, Amikacin, Clarithromycin, and Trimethroprim/Sulfamethoxazole. However, the abscess volume increased reaching 10 cm × 10 cm with undefined limits and skin erosion; the patient had an intermittent low-grade fever (38.0–38.5°C) with one peak each day for the next 3 months, and she received several courses of antibiotic without improvement. Then, the patient was admitted to the hospital because general illness, including fever, and tuberculosis infection was suspected. After hospital admission, an excisional clavicular biopsy reported inflammation and abundant AFB (10 bacilli per field, in 20 fields) in the soft tissue (Figures 1A,B) and osteomyelitis (Figure 1C). Computerized axial tomography showed evidence of multiple infectious abscesses (Figure 1D). PCR analysis of material from the supraclavicular biopsy was positive for Mycobacterium tuberculosis complex and the Gene-X-pert test was positive for M. tuberculosis sensitive to rifampin. A diagnosis of disseminated tuberculosis was made, and the patient received anti-mycobacterial treatment with Rifampin, Isoniazid, Pirazynamide, and Ethambutol at conventional doses. A repeat biopsy of supraclavicular abscess showed nine AFB in 100 fields; with this improvement, the patient was discharged from the hospital on continued anti-mycobacterial treatment with Rifampin plus Isoniazid for18 months, with good clinical evolution.

The patient had neutropenia and lymphopenia during infection episodes; serum IgA levels were transiently low in several assessments, returning to normal values after recovering from active infections. Values for IgG, IgM, and IgE were normal. The patient was diagnosed with chronic hepatitis, with high values of alanine-aminotransferase, aspartate-aminotransferase, and gamma-glutamyltranspeptidase, most likely due to anti-mycobacterial and anti-fungal treatments, since a liver biopsy showed mild chronic hepatitis, without fibrosis or copper deposits. Additional tests for liver function were normal. Serology tests for viral infections (including hepatitis A, B, and C, CMV, HIV, and EBV) were all negatives. Tests for autoantibodies against DNA, cardiolipin, beta-2 glycoprotein, endomysium, and smooth muscle were all negatives.

We found a relatively low production of interferon gamma (IFN-γ) in response to BCG and BCG + IL-12 treatment of diluted whole blood in the patient compared to healthy controls (BCG = 891 pg/mL vs BCG + IL-12 = 5,025 pg/mL for patient, compared to nine healthy controls: GeoMean ± SEM with BCG = 1,369 ± 1,878 and with BCG + IL-12 = 9,579 ± 1,935 pg/mL). The IL-12Rβ1 expression levels on PHA-T cell blasts by flow cytometry were normal in the patient (data not shown). In vitro responses to IFN-γ showed an increased production of IL-12p70 in the patient compared to healthy controls (Figure 2A), upon BCG and BCG + IFN-γ stimulation, suggesting an alteration in the IFN-γ receptor or downstream signaling. Membrane expression of IFN-γ receptor 1 (CD119) on patient’s CD14+ cells was similar to healthy controls (data not shown).

Signal transduction and activator of transcription 1 (STAT1) showed an increased tyrosine phosphorylation at position 701 in CD14+ cells in PBMCs from the patient stimulated with IFN-γ compared to controls, as assessed by flow cytometry (Figure 2B, histograms); similar STAT1 hyperphosphorylation was found in Epstein-Barr virus lymphoblastoid cell lines (EBV-LCLs) from the patient (Figure 2B, Western blot), suggesting a gain-of-function (GOF) mutation in STAT1. Since previous studies demonstrated the involvement of impaired dephosphorylation for other GOF mutations in STAT1 (1–3), we characterized the inhibition of phosphorylation by staurosporine in EVB-LCLs. We found resistance to staurosporine-mediated inhibition of phosphorylation of STAT1 in the patient than in healthy control (Figure 2C).

Sequencing of the STAT1 gene in PBMCs showed a mutation in the DNA-binding domain (c.1154 C>T, p.T385M) only in the patient (absent in the parents and brother), indicating a de novo mutation (Figure 3A). Threonine 385 is a highly conserved amino acid in several vertebrate species, representing a critical amino acid for STAT1 function; this mutation has already been shown to cause STAT1 GOF (4). Recently, the use of the Jak kinase inhibitor Ruxolitinib was demonstrated to correct STAT1 hyperphosphorylation and ameliorate Candida infection (5, 6); therefore, we tested and demonstrated inhibition of STAT1 phosphorylation by Ruxolitinib in EBV-LCLs from our patient upon stimulation with IFN-α and IFN-γ (Figure 3B).

Disseminated mycobacterial infections that occur in children are usually associated with primary immunodeficiency and are frequently related to impaired IFN-γ-dependent immune responses. Children affected with the Mendelian susceptibility to mycobacterial diseases show variable susceptibility to mycobacterial infections ranging from weakly virulent species, such as Mycobacterium bovis BCG and Mycobacterium avium, to the more virulent M. tuberculosis; these patients have mutations in genes controlling the IFN-γ production (IL12B, IL12RB1, IRF8, ISG15, and NEMO) or in genes involved in the response to IFN-γ (IFNGR1, IFNGR2, STAT1, IRF8, and CYBB) (7). These genes encode distinct proteins participating in the IFN-γ/IL-12 circuit, which is crucial for the immune response against mycobacteria in humans (7–10).

Engagement of IFN-γ by its receptor in macrophages activates JAK1 and JAK2 intracellular kinases, which auto- and trans-phosphorylate themselves. They phosphorylate tyrosine (Y) 440 on the IFN-γ receptor 1 creating docking sites for STAT1 coupling, which in turn is phosphorylated at Tyrosine 701 (pY701) by the same JAKs. Once STAT1 is phosphorylated, it forms homodimers in the cytoplasm; these homodimers are called GAFs or Gamma-activated factors, which translocate to the nucleus where they bind to specific DNA sequences called GASs or Gamma-activated sequences. GAFs are responsible for IFN-γ-dependent gene induction, enabling the macrophage to perform intracellular killing of phagocytized microbes (10, 11). STAT1 also forms heterodimers with STAT2 upon IFN-α or IFN-β activation mediated by their respective receptors; these STAT1/STAT2 heterodimers bind to IRF9 to form the mature transcription factor ISGF3, which binds to the specific DNA sequence ISRE and induces transcription of genes that participate in the antiviral immune response (12).

Mutations in STAT1 in humans have previously been identified. Recessive loss-of-function (LOF) mutations in STAT1 cause reduced signaling involving the STAT1 transcription factor, affecting immunity to intracellular pathogens primarily, like mycobacteria and viral infections due to impaired IFN-γ and IFN-α signaling, respectively (13). Dominant LOF mutations in STAT1 impair its phosphorylation or DNA-binding activity and are associated with mycobacterial disease due to a specific impairment in IFN-γ signaling (14). In contrast, GOF mutations in STAT1 cause an increased phosphorylation of Y701 of STAT1 and a reduced production of IL-17 by T cells from affected persons; these mutations have been found in patients with fungal diseases, primarily caused by Candida spp. as well as by other fungal diseases such as Coccidioidomicosis and Histoplasmosis (1, 15, 16). Chronic mucocutaneous candidiasis (CMC) occurs with persistent or recurrent infections with Candida spp. in the skin, nails, oral, or genital mucosa; approximately half of all cases studied and genetically characterized worldwide are due to GOF STAT1 mutations, and the other half are due to mutations in other genes that impair the IL-17-mediated immune response, such as STAT3, IL17RA, IL17F, IL17RA, IL17RC, ACT1, RORC, IL12RB1, AIRE, and CARD9 (17–23).

In 2016, an international multicenter study (24) examined 274 patients with GOF mutations in STAT1; while 268 patients had CMC only 17 patients had mycobacterial infections (6% of the studied patients). This is paradoxical and unexplained, as LOF mutations in STAT1 are well-known causes of mycobacterial disease (25).

We identified a GOF mutation in STAT1 in our patient with confirmed M. tuberculosis disseminated infection and CMC. The mutation found in the patient increased the phosphorylation of Y701 in the STAT1 protein, which was proved to be resistant to Staurosporine, but sensitive to Ruxolitinib, which has previously been used against CMC in two clinically distinct patients (5, 6). This suggests an alternative treatment for infections in this type of patient; however, its effects in the elimination of mycobacterial infections remain to be determined.

Chronic mucocutaneous candidiasis is the most frequent infection associated to STAT1 GOF mutations (1, 16), although patients have also bacterial and viral infections (24). Interestingly, Sampaio et al. (16) identified a patient with the mutation T385M (the same found in our patient) and a sporadic infection with Mycobacterium fortuitum, indicating that there is also susceptibility to Mycobacterial infection with the STAT1 GOF mutation. The mycobacterial infections found in 17 out of 268 patients with heterozygous STAT1 GOF mutations recently reported by Toubiana et al. demonstrate the susceptibility to these infections (24).

In the patient reported here, BCG vaccination caused an initial mycobacterial infection during her first year of life and debridement of the axillar abscess relieved infection symptoms. Although AFB were found in gastric juices from patient at 3 years old, isolation of mycobacteria was unsuccessful. Later, the infection in the clavicule showed AFB in the bone and soft tissue and the Gene-X-pert-positive result demonstrated an M. tuberculosis infection, with clinical symptoms of dissemination (see Figure 1). Thus, clinical phenotype and laboratory testing indicated that the patient had a bona fide disseminated mycobacterial infection. Recently, Baris et al. (26) observed a pulmonary M. tuberculosis infection in a patient with the same T385M GOF mutation in STAT1, while Kataoka et al. (27) identified a patient with an R274G STAT1 GOF mutation and disseminated M. tuberculosis infection albeit with negative Quantiferon and tuberculin skin tests. Similarly, PPD testing was negative in our patient at 3 and 6 years old, despite her mycobacterial infections, indicating a defective T-cell response.

The mechanism underlying STAT1 GOF mutations and susceptibility to mycobacterial infection is not well understood, but cytokine production is altered in leukocytes. Sampaio et al. (16) found increased production of IL-12 as well as chemokines CXCL9 and CXCL10 in response to IFN-γ in patients with STAT1 GOF mutations. Similarly, we found an increased production of IL-12 (shown) and CXCL10 (not shown) in whole blood from the patient stimulated with BCG compared to healthy controls. IFN-γ is thought to be an essential cytokine necessary to control of mycobacterial infections. However, we found by ELISA only 35% less IFN-γ production in whole blood from the patient stimulated with BCG compared to healthy controls; by flow cytometry, we observed diminished IFN-γ producing T cells in PBMCs from the patient compared to control (29 vs 42%) but almost absent in IL-17A-producing T cells in the patient compared to control (0.2 vs 1.6%), upon PMA + ionomycin stimulation. Liu et al. (1) found a diminished (albeit not significant) percentage of IFN-γ-producing cells in patients with STAT1 GOF mutations compared to healthy controls and significant lower proportion of IL-17-producing T cells (by flow cytometry) similar to other authors (2, 16, 28). Therefore, consistently, there is a significant reduction in IL-17 production but only partial IFN-γ reduction in patients with STAT1 GOF mutations. In a murine model of lung infection with BCG, IL-17-deficient mice showed impaired Th1-IFN-γ production and defective granuloma formation compared to wild-type mice (29), suggesting a protective role of IL-17 in vivo. However, human patients lacking the IL-17A receptor do not suffer from mycobacterial infections (20), whereas patients with mutations in RORC lack the Th1* subset able to produce both IFN-γ and IL-17 (patients do not produce IL-17 A and IL-17 F) and suffer from both Mycobacterium and Candida infections (23). Mycobacteria induce both Th1 and Th17 responses. T cells from children vaccinated with BCG stimulated in vitro with PPD produce high amounts of both IFN-γ and IL-17 compared to non-vaccinated children [reviewed in Ref. (30)]. Interestingly, IL-17 production is reduced in patients with active tuberculosis and its production increases when tuberculosis symptoms are ameliorated after drug treatment, to levels found in healthy controls (31–33), suggesting that IL-17 may participate for the control of mycobacterial infections. Nevertheless, the role of Th17 cells in immune responses against tuberculosis is controversial because there appears to be distinct roles in latent and active human infections, while in animal models, its effect can be beneficial or detrimental (34). Other studies suggest that a high production of Type I IFNs and IL-10 are detrimental during active tuberculosis, inhibiting the protective effects of IFN-γ (35–37). IFN-α has demonstrated beneficial effects as adjuvant during pharmacological treatment of tuberculosis in humans, but paradoxically hepatitis treatment with IFN-α reactivates latent tuberculosis (34). In contrast, Uzel and Holland (38) found a diminished production of IL-10 in response to PHA in PBMC from three patients with STAT1 GOF mutations compared to healthy controls, suggesting that an increased production of IL-10 may not occur in patients with these mutations. The pathway of type I IFNs production and the consequent activated genes has not been studied in patients with STAT1 GOF mutations and mycobacterial infections and need to be addressed in further studies.

We described here a patient with a bona fide M. tuberculosis disseminated infection, related to a GOF mutation in STAT1, a genetic change associated primarily with CMC. GOF mutations in STAT1 affect IL-17 production, and IFN-γ to a lesser extent. More research is required to investigate the mechanisms associated with IFN-γ and IL-17 cytokine production, distinct types of STAT1 mutations, and the resulting susceptibility to different infections.

This study was performed in accordance with the recommendations of the committee of ethics in research of the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubiran (INCMNSZ). All participants and parents of the patient gave written informed consent in accordance with the Declaration of Helsinki. These studies were approved by the Institutional Review Board of INCMNSZ, protocol code BQO-751-12-16-1.

SPS designed the experiments, performed flow cytometry experiments and wrote the paper. JLLF provided medical care to the patient, contributed to medical figures and wrote the clinical description of the case. YG performed the experiments (flow cytometry and sequencing) and collaborated on writing of the paper. LMR and MLVA maintained cell cultures, performed ELISA and Western blot experiments, contributed to figures and participated in manuscript writing. SSP provided the pathology reports and contribute to the pathology figures. MNF and SME provided medical attention to the patient and contributed to the paper writing. JLC, AP and SBD collaborated in the design of the studies, sequencing to determine the mutation and participated in writing the paper MT coordinated the work, edited the manuscript and wrote the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.