Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency

Abstract

ISG15 is an interferon (IFN)-α/β-inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes-granulocyte, in particular-reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ-inducing secreted molecule for optimal antimycobacterial immunity.

Fig. 1

The familial segregation and expression pattern of the ISG15 allele indicates recessive inheritance and an absence of protein production. Familial segregation in a family from Turkey (Kindred A) and a family from Iran (Kindred B) (A). Graphical representation of the proISG15 protein. The LRLRGG ISGylation domain, the 8-amino acid sequence (black) cleaved to yield active ISG15, and the putative proteins synthesized in the patients (B) are shown. EBV-B cells from Control 1 (C1), Control 2 (C2), a STAT1^−/− patient (negative control) and patient 1 (P1), were left untreated or treated with IFN-α. The cells were then lysed and the lysates were subjected to western blotting (C). EBV-B cells from Control 1 (C1), a STAT1^−/− patient and patient 1 (P1) were stained with ISG15 and STAT1 antibodies and analyzed by flow cytometry (D). SV-40-fibroblasts from Control 1 (C1), a STAT1^−/− patient, P1, and P2 were analyzed as in C (E). All experiments presented were performed at least 3 times.

Fig. 2

ISGylation and viral susceptibility in cell lines derived from patients with mutations in ISG15. Control SV-40-immortalized fibroblasts (C1), fibroblasts derived from P1, P2 or a STAT1^−/− patient were either left untreated or treated with IFN-β for 24 hours. Cell extracts were analyzed by SDS-PAGE and immunoblotting with antibodies against ISG15, IFIT3 or tubulin (A). SV-40 fibroblast cell lines from patients P1 and P2 were mock-transfected (M), transfected with a plasmid encoding 3XFLAG-ISG15 (WT) or transfected with a plasmid encoding a form of ISG15 unable to conjugate with proteins (AA). Eighteen hours after transfection, we treated the cells with IFN-β for an additional 18 hours. Cell extracts were analyzed by SDS-PAGE and immunoblotting with FLAG, IFIT3 or tubulin antibodies (B) (A and B are representative of at least 3 independent experiments ). HSV-1 replication was monitored by assessing the fluorescence of GFP fused to a viral capsid protein, in SV40-fibroblasts from a healthy control C1, P1, P2 and a STAT1^−/− patient, infected with HSV-1 at a multiplicity of infection (MOI) of 0.2 for the times indicated. Cells were treated with either medium alone (C) or with IFN-α (D) for 24 h before infection. The results shown are the means of four independent experiments. Error bars indicate the SEM.

Fig. 3

ISG15 secretion and the induction of IFN-γ production in leukocytes. Control and P1 leukocytes were left unstimulated or were stimulated with BCG and IFN-α2b. After 0 h and 12 h, cells and supernatants were harvested and subjected to western blotting (A) or TNF-α ELISA (B). Resting control and P1 leukocytes were labeled extracellularly with CD16 and CD3 antibodies and intracellularly with ISG15 antibody and were subjected to ImageStreamX analysis with bright field (BF) and side scatter (SSC) also shown (C). IFN-γ secretion was measured by ELISA in PBMCs stimulated with vehicle (−), various doses of recombinant human ISG15 (including boiled recombinant human ISG15 to exclude LPS contamination) (D), recombinant human ISG15 and IL-12, alone and in combination (E). The IFN-γ secretion results shown are representative means of at least 3 independent experiments. Error bars indicate the SEM.

Fig. 4

Impaired IFN-γ production in ISG15^−/− patients and rescue by exogenous ISG15. Cytokine production in the supernatants of whole-blood cells from local controls (n=29), travel controls (n=9), ISG15^−/− (n=3) and IL12RB1^−/− patients (n= 58), left unstimulated or stimulated with BCG alone or BCG plus cytokine (indicated), as detected by ELISA (A to C); alternatively, recombinant human ISG15 was added at the same time or 24 h before whole-blood activation by BCG and IL-12 (D). The amounts of cytokine secreted are normalized for 10⁶ PBMC on a logarithmic scale, and medians are indicated by solid bars. Differences in log-transformed IFN-γ levels after stimulation with BCG + IL-12 were assessed 1) between ISG15^−/− subjects and travel controls in Student's t-test (A), and 2) between ISG15^−/− subjects before and after adding recombinant human ISG15 by two-way analysis of variance, to take into account both activation by recombinant ISG15 and activation time (D). IFN-γ secretion was measured in whole blood stimulated with vehicle (−), BCG or BCG plus IL-12, in the presence of vehicle (−), a blocking antibody against ISG15, or an IgG1 isotype control (E).