A 2.5-year-old female (P1) was admitted to the hospital because of 3 weeks of daily fever (over 38 C) associated with fatigue, vomiting and diarrhea. She was born in Italy from Pakistani non-consanguineous parents; her past medical history was uneventful, and the family history was unremarkable. She had not received BCG vaccine, nor had never been to Pakistan. At admission, her blood tests revealed microcytic anemia (Hb 6.5 g/dl, MCV 60.9 fl, RDW 19.4%), raised C-reactive protein (CRP 125 mg/L, range <5 mg/L) and procalcitonin (9.2 ng/ml, range <0.1 ng/ml), leukocytosis (WBC 21,600 cells/mmc) with neutrophilia (ANC 17,170 cells/mmc). She had normal lymphocyte count (ALC 3,230 cells/mmc), and monocyte count at the lower range (AMC 230 cells/mmc). On physical examination, she had mild spleen enlargement. The abdomen ultrasound and the computed tomography-scan revealed a spleen length of 8 cm [mean of normal age-related spleen size from 6.8 to 7.5 cm ] with multiple dishomogenous areas, slight hepatomegaly, without focal lesions, and multiple mediastinal and mesenteric lymph nodes showing internal colliquation. The infectious disease screening included blood cultures, Brucella, Rickettsia, Bartonella, Coxiella, Leishmania, Toxoplasma, Plasmodium, Herpesviruses, Epstein-Barr virus, and HIV serology, all tested negative. The Mantoux tuberculin skin test read after 48 h resulted on induration of 5 mm, while the QuantiFERON-TB Gold tested negative. She underwent a laparoscopic biopsy of an abdomen lymph node (20 x 14 mm) that showed a necrotizing granulomatous lymphadenitis with focal colliquation and the Mycobacterium avium was detected by PCR assay. The patient was commenced with the association therapy of clarithromycin, ethambutol and rifabutin, the latter discontinued after 6 months because of an acute drug-related uveitis. The dual therapy with clarithromycin and ethambutol was continued for 18 months. The patient fully recovered. At onset, routine immunological tests revealed slightly elevated levels of serum immunoglobulin (Ig) M (IgG 1,220 mg/dl -range 462-1,710 mg/dl-, IgA 167 mg/dl -range 27-173 mg/dl-, IgM 465 mg/dl -range 62-257 mg/dl-), and IgE (575 range KU/L -range <140 KU/L-), normal neutrophil respiratory burst assay, and normal bone marrow aspirate. The analysis of the lymphocytes subsets displayed a prevalence of effector T and B cells, an increased proportion of activated HLA-DR+ T cells, and reduction of NK cell percentages (CD16+CD56+ 2.7%/90 cells/mmc, normal range 5-28.4%/100-1,000 cells/mmc) (Table 1).
IFNGR1 gene sequencing revealed a heterozygous small deletion in the exon 6, the c.819_822delTAAT (p.N274Hfs*2). This mutation is already reported as responsible of AD partial IFNgammaR1 deficiency. The flow cytometry analysis of IFNgammaR1 expression on patient's monocytes showed that the receptor was present at a higher level on cell's surface (Figures 1A,B), while STAT1 phosphorylation (pY-701) following stimulation with IFNgamma was markedly reduced, as compared to the healthy control (Figures 1C,D).
Analysis of DCs was performed based on specific cell surface markers. Myeloid/classical DCs (mDCs) express typical myeloid antigens (CD11c, CD13, CD33, CD11b) with the majority of them expressing CD1c/BDCA1, while plasmacytoid DCs (pDCs) express CD123, CD303/BDCA2, CD304/BDCA4, and produces IFNalpha after activation with CpG or viral infection. At the time of P1 mycobacterial infection diagnosis, we observed a marked reduction of pDCs equal to 0.04%/143 cells/mmc (normal range 0.16-0.76%) and of mDCs equal to 0.05%/179 cells/mmc (normal range 0.18-0.92%) (Table 1). Monitoring of DCs subsets during the infection treatment showed that the percentage of DCs progressively increased, reverting to the normal levels by the end of the treatment (18 months) and remaining normal in the following months (Figure 2A). In addition, ELISA measurements of IFNalpha and CXCL10 in 24-h supernatants of PBMCs incubated with CpG or with increasing copies of human Herpes Simplex Virus Type 1 (HSV1) demonstrated lower levels of these cytokines in P1 as compared to the healthy control (Figures 2B,C), which is in accord with the blood depletion of pDCs.
Next, we compared DCs subsets in other two patients (P2 and P3) with AD partial IFNgammaR1 deficiency, in P2 during a mycobacterial infection, while P3 was free of acute mycobacterial infection at the time the blood sample was collected. We also analyzed DCs in three other patients that had a mycobacterial cervical lymphadenopathy but still without any evidence of genetic mutations associated with MSMD. We detected a low number of both pDCs and mDCs subsets in P2, while DCs counts were normal in all the other investigated patients (Figure 2D). Finally, in P1, we studied DCs maturation by flow cytometry: the analysis of HLA-DR, CD80, CD83, and CD86 markers revealed that, after 24 h of in vitro culture with LPS, DCs exhibited a normal pattern of maturation (Figure 3).
In P1, the analysis of the lymph node biopsy revealed a granulomatous necrotizing lymphadenitis with BDCA2+ plasmacytoid dendritic cells reduction (Figures 4A-E), as compared with other age-matched granulomatous lymphadenitis of mycobacterial origin derived from control subjects (Figures 4F-L); the distribution of CD20+ B-cells and CD3+ T-cells of the nodal parenchyma was normal.