Periodic Reporting for period 1 - REI_MSMD (Inborn errors of translation reinitiation in humans with Mendelian susceptibility to mycobacterial disease)
Berichtszeitraum: 2023-09-01 bis 2025-08-31
The clinical outcome of any infection varies considerably between individuals, ranging from silent infection in most, to lethal disease in a few. Tuberculosis (TB), caused by Mycobacterium tuberculosis, has been a deadly infectious disease, with at least one billion deaths in the last 2,000 years. However, infection with M. tuberculosis is silent or benign in >90% of infected individuals. Forward genetics studies of rare patients with clinical disease due to the weakly virulent Bacillus Calmette–Guérin (BCG) live-attenuated vaccine against TB, or atypical environmental mycobacteria (EM) led to the discovery of human genetic and immunological determinants of TB. Severe disease due to BCG or EM in otherwise healthy individuals, particularly those without HIV infection or immunosuppression, is referred to as Mendelian susceptibility to mycobacterial disease (MSMD) because of the frequent occurrence of multiplex families and parental consanguinity. MSMD is relatively rare, occurring in ~1 in 50,000 individuals, and is typically ‘isolated’ (~75% of cases) or, rarely, ‘syndromic’ (~25%). Patients with isolated MSMD are otherwise healthy and normally resistant to most common microbes, except, occasionally, other intramacrophagic pathogens, including some bacteria (e.g. Salmonella), fungi (e.g. Coccidioidomyces), and parasites (e.g. Leishmania).
Over the last 25 years, the discovery of inborn errors of interferon-gamma (IFN-) immunity has defined the root cause and immunological mechanism of MSMD and TB. With one possible exception (ZNFX1 deficiency), all genetic defects underlying both isolated and syndromic MSMD perturb IFN- immunity (germline mutations of genes encoding IFN-R1, IFN-R2, IFN-, STAT1, IRF1, JAK1, IL-12Rß1, IL-12Rß2, IL-23R, IL-12p40, TYK2, SPPL2A, IRF8, NEMO, CYBB, ISG15, USP18, RORT, and T-bet). Many genetic defects directly impair the production of IFN-, or the response to this cytokine (IFN-R1, IFN-R2, JAK1, STAT1, IRF1), whereas others affect the production of, or response to the IFN--inducing cytokines IL-12, IL-23, and ISG15 (IL-12Rß1, IL-12Rß2, IL-23R, IL-12p40, TYK2, ISG15). Deficiencies of the IL-12-specific IL-12Rß230 and IL-23-specific IL-23R also underlie MSMD, suggesting that neither of these two IFN--inducing cytokines is dispensable for antimycobacterial immunity.
Human genetic studies of MSMD have, thus, revealed that IL-12- and IL-23-dependent IFN-γ immunity is essential for host defense against weakly virulent mycobacteria. Upon infection with mycobacteria, macrophages produce IL-12 and IL-23, which stimulate natural killer (NK) and T lymphocytes to produce IFN-. Several lymphocyte subsets have been identified as critical producers of IFN- and mediators of antimycobacterial immunity: patients with autosomal recessive (AR) SPPL2A or RORT deficiency, or with autosomal dominant (AD) IRF8 deficiency suffer from MSMD due to a lack of T-helper (TH) 1* cells, whereas AR T-bet deficiency impairs the development and IFN- production of NK cells, innate-like adaptive lymphocyte subsets (mucosal-associated invariant T cells [MAIT], V2+ T, and invariant natural killer [iNKT] cells), and TH1 cells. In IL-12Rß1-, IL-12Rß2-, and IL-23R-deficient patients, these subsets (NK, MAIT, V2+ T, iNKT, TH1 and TH1*) also fail to produce IFN- in response to IL-12 and/or IL-23. IFN- can stimulate most cell types, but is crucial for the killing of mycobacteria within phagocytes, and it also boosts the production of IL-12 and IL-23 by these cells, forming a positive feedback loop.
Molecular genetics studies of MSMD have revealed the mechanisms of antimycobacterial immunity in humans in natura, demonstrating that IFN- acts as a key antimycobacterial cytokine rather than an antiviral interferon. Severe viral diseases are rare in patients with MSMD, occurring mostly in patients with syndromic MSMD who also have impaired IFN-/ immunity (mutations of JAK1, STAT1, TYK2). Moreover, the study of MSMD has led to the discovery of both rare and common determinants of clinical TB, which is caused by M. tuberculosis, a bacterium 1,000 times more virulent than those implicated in MSMD2. Most genetic etiologies of MSMD have incomplete penetrance for MSMD and can manifest as genetic etiologies of TB. Moreover, homozygosity for the common P1104A allele of TYK2 selectively impairs the IL-23-dependent induction of IFN- and accounts for about 1% of cases of TB in patients of European descent. The study of MSMD has, therefore, had both biological and medical implications. However, a clear genetic etiology has yet to be found for about half the MSMD patients identified to date, implying that our understanding of antimycobacterial immunity is incomplete. In this context, we searched for new genetic causes and immunological mechanisms of MSMD.
Over the last 25 years, the discovery of inborn errors of interferon-gamma (IFN-) immunity has defined the root cause and immunological mechanism of MSMD and TB. With one possible exception (ZNFX1 deficiency), all genetic defects underlying both isolated and syndromic MSMD perturb IFN- immunity (germline mutations of genes encoding IFN-R1, IFN-R2, IFN-, STAT1, IRF1, JAK1, IL-12Rß1, IL-12Rß2, IL-23R, IL-12p40, TYK2, SPPL2A, IRF8, NEMO, CYBB, ISG15, USP18, RORT, and T-bet). Many genetic defects directly impair the production of IFN-, or the response to this cytokine (IFN-R1, IFN-R2, JAK1, STAT1, IRF1), whereas others affect the production of, or response to the IFN--inducing cytokines IL-12, IL-23, and ISG15 (IL-12Rß1, IL-12Rß2, IL-23R, IL-12p40, TYK2, ISG15). Deficiencies of the IL-12-specific IL-12Rß230 and IL-23-specific IL-23R also underlie MSMD, suggesting that neither of these two IFN--inducing cytokines is dispensable for antimycobacterial immunity.
Human genetic studies of MSMD have, thus, revealed that IL-12- and IL-23-dependent IFN-γ immunity is essential for host defense against weakly virulent mycobacteria. Upon infection with mycobacteria, macrophages produce IL-12 and IL-23, which stimulate natural killer (NK) and T lymphocytes to produce IFN-. Several lymphocyte subsets have been identified as critical producers of IFN- and mediators of antimycobacterial immunity: patients with autosomal recessive (AR) SPPL2A or RORT deficiency, or with autosomal dominant (AD) IRF8 deficiency suffer from MSMD due to a lack of T-helper (TH) 1* cells, whereas AR T-bet deficiency impairs the development and IFN- production of NK cells, innate-like adaptive lymphocyte subsets (mucosal-associated invariant T cells [MAIT], V2+ T, and invariant natural killer [iNKT] cells), and TH1 cells. In IL-12Rß1-, IL-12Rß2-, and IL-23R-deficient patients, these subsets (NK, MAIT, V2+ T, iNKT, TH1 and TH1*) also fail to produce IFN- in response to IL-12 and/or IL-23. IFN- can stimulate most cell types, but is crucial for the killing of mycobacteria within phagocytes, and it also boosts the production of IL-12 and IL-23 by these cells, forming a positive feedback loop.
Molecular genetics studies of MSMD have revealed the mechanisms of antimycobacterial immunity in humans in natura, demonstrating that IFN- acts as a key antimycobacterial cytokine rather than an antiviral interferon. Severe viral diseases are rare in patients with MSMD, occurring mostly in patients with syndromic MSMD who also have impaired IFN-/ immunity (mutations of JAK1, STAT1, TYK2). Moreover, the study of MSMD has led to the discovery of both rare and common determinants of clinical TB, which is caused by M. tuberculosis, a bacterium 1,000 times more virulent than those implicated in MSMD2. Most genetic etiologies of MSMD have incomplete penetrance for MSMD and can manifest as genetic etiologies of TB. Moreover, homozygosity for the common P1104A allele of TYK2 selectively impairs the IL-23-dependent induction of IFN- and accounts for about 1% of cases of TB in patients of European descent. The study of MSMD has, therefore, had both biological and medical implications. However, a clear genetic etiology has yet to be found for about half the MSMD patients identified to date, implying that our understanding of antimycobacterial immunity is incomplete. In this context, we searched for new genetic causes and immunological mechanisms of MSMD.
We report X-linked recessive MCTS1 deficiency in men with mycobacterial disease from kindreds of different ancestries (from China, Finland, Iran, and Saudi Arabia). Complete deficiency of this translation re-initiation factor impairs the translation of a subset of proteins, including the kinase JAK2 in all cell types tested, including T lymphocytes and phagocytes. JAK2 expression is sufficiently low to impair cellular responses to IL-23 and partially IL-12, but not other JAK2-dependent cytokines. Defective responses to IL-23 preferentially impair the production of IFN- by innate-like adaptive MAIT and T lymphocytes upon mycobacterial challenge. Surprisingly, the lack of MCTS1-dependent translation re-initiation and ribosome recycling seems to be otherwise physiologically redundant in these patients. These findings suggest that X-linked recessive human MCTS1 deficiency underlies isolated mycobacterial disease by impairing JAK2 translation in innate-like adaptive T lymphocytes, thereby impairing the IL-23-dependent induction of IFN-.
We describe a surprising, new genetic etiology of MSMD: XR MCTS1 deficiency in six male individuals from five unrelated kindreds and four distant ancestries. Complete MCTS1 deficiency impairs ribosome recycling and translation re-initiation in the patients’ cells. JAK2 is one of about 150 human genes subject to MCTS1-mediated translation re-initiation. JAK2 protein levels in MCTS1-deficient myeloid and lymphoid cells are about three- to four-fold lower than those in control cells, with a greater decrease in levels observed in lymphoid than in myeloid cells. Responses to IFN-γ and IL-12, both of which normally require JAK2, were normal in these cells. By contrast, the response to IL-23 was impaired in the patients’ T-cell blasts and MCTS1 KO HEK Blue cells, which was rescued by exogenous expression of MCTS1 or JAK2. The patients MAIT and V γ T-lymphocyte subsets produce only small amounts of IFN-γ in response to IL-23 and BCG. We show here that decreases in JAK2 levels or activity impair IL-23-dependent IFN-γ production by lymphocytes, providing a molecular and cellular basis for disease, and mechanistically and causally connecting the MCTS1 genotype and the MSMD phenotype. Remarkably, an unbiased genome-wide forward genetics approach revealed an unexpected MSMD phenotype in patients with human MCTS1 deficiency.
Our finding is unexpected because MCTS1 is ubiquitously expressed and has a fundamental molecular function in the dissociation of deacylated tRNAs from post-termination 40S ribosomal complexes during ribosome recycling (on mORF stop codons) and re-initiation (on uORF stop codons). The translation re-initiation functions of DENR and MCTS1 are essentially identical in amplitude 58,59,62. However, DENRKO is embryonically lethal in mice, whereas MCTS1KO mice are viable 60. This is consistent with the more severe ribosome recycling defect of DENRKO HeLa cells than of MCTS1KO HeLa cells. Furthermore, we found no MSMD patient homozygous for rare candidate DENR variants. The apparent lack or extreme rarity of DENR-deficient patients suggests that this deficiency may be embryonically lethal in humans, as it is in mice. This contrasts with our findings that all five patients with MCTS1 deficiency have isolated MSMD and are otherwise healthy at ages of six months to 18 years. However, only one MCTS1-deficient patient has yet reached adulthood, and other clinical phenotypes may appear as the patients age.
We define the set of human MCTS1-dependent genes in cells from MCTS1-deficient patients. We identify JAK2 as a translational target of DENR-MCTS1 and probably the key contributor to MSMD. Decreases in the levels of other MCTS1-dependent proteins do not appear to have clinical consequences. Surprisingly, partial JAK2 deficiency impairs cellular responses to IL-23 and the subsequent induction of IFN-γ in lymphocytes, but not other cytokine response pathways involving JAK2. DENRKO has been reported to impair JAK2 translation and subsequent responses to IFN- in mouse tumor cells85. Responses to IL-12 and IL-23 were not tested in this previous study. We found the same connection between MCTS1 and JAK2 in human cells, but the response to IFN- was intact. Low levels of JAK2 may have a much greater effect in IL-23- and IL-12-responsive cells than in IFN--responsive cells. It is probably no coincidence that the four types of MSMD-causing TYK2 variants, including the common P1104A variant, have an impact on the IL-23 response pathway, whereas only two of these types of variants also impair responses to IL-1235. This finding suggests that the IL-23R is more dependent on qualitative and quantitative variations of JAK2 or TYK2 than the IL-12R. Biochemical differences may be responsible for the weaker IL-23R signaling observed.
Our findings support our previous reports that human IL-23 is a key IFN--inducing cytokine18,30,42,73. Despite having the p40 chain in common, IL-23 has historically been seen as an IL-17-inducing cytokine73,88-91, whereas IL-12 is seen as the key IFN--inducing cytokine73,91. Recent discoveries have suggested that human IL-23 plays a key role in IFN--mediated immunity to mycobacteria, and a more minor role in IL-17-mediated immunity to fungi. First, AR complete IL-23R deficiency underlies MSMD with almost complete clinical penetrance but only a mild form of CMC with incomplete penetrance30,42. Second, homozygosity for the common TYK2 P1104A allele selectively impairs responses to IL-23 and underlies MSMD with low penetrance and tuberculosis with high penetrance, but has no effect on susceptibility to CMC18,54. Third, impairment of the cellular response to IL-23 is the only mechanism underlying mycobacterial disease common to patients with the five forms of AR TYK2 deficiency28,60. Fourth, MCTS1-deficient patients have impaired cellular responses to IL-23 and isolated MSMD, with no detectable IL-17 deficiency or CMC. Collectively, these observations suggest that human IL-23 acts more as an IFN-inducing antimycobacterial cytokine than as an IL-17-inducing antifungal cytokine.
We found that IFN- induction in response to IL-23 stimulation was impaired in the V2+ T and MAIT cells of MCTS1-deficient patients. This finding is consistent with previous reports, as V2+ T and MAIT cells are among the subsets producing the largest amounts of IFN- in response to IL-2330,42. Furthermore, the IFN- production of these cells in response to IL-23 is impaired in IL-12R1-/-, IL-23R-/- and TYK2-/- individuals30,42,60. NK cells are also potent inducers of IFN- upon stimulation with IL-23, but their transcriptional response to IL-23 is only moderately impaired in MCTS1-deficient patients. Interestingly, the induction of IFN- was also impaired in the patients’ TH1* cells. TH1* cells have a lower level of IFN- induction than MAIT, V2+ T and NK cells. Nonetheless, TH1* cells display more than an eight-fold induction of IFN- in response to IL-23 (Figure S8F), this induction being totally abolished in MCTS1-, IL-12R1-, and IL-23R-deficient patients. TH1* cells are the major lymphocyte subset producing IFN- after exposure to BCG in vitro92,93. The counts of these cells in the blood increase in individuals with latent tuberculosis infection94. IL-23 may, thus, also contribute to TH1*-mediated immunity to mycobacteria.
Finally, we can speculate about the clinical phenotype of MCTS1-deficient patients in the absence of BCG vaccination. Surprisingly, none of the six MCTS1-deficient individuals had EM disease. This may be due to the protection afforded by BCG disease, as seen in IL-12Rß1-deficient patients38,39. It may also reflect an incomplete IFN- deficit, as only one of the seven reported IL-23R-deficient patients suffered from EM disease41,42 and cellular responses to IL-23 are impaired, but not abolished in MCTS1-deficient patients. In regions of endemic tuberculosis, in which BCG vaccination is not widely or efficiently performed, or if penetrance for BCG disease is incomplete, MCTS1 deficiency may be revealed by severe tuberculosis. Overall, this study reveals a surprising mechanistic connection between deficiency of a basic, ubiquitous, biochemical mechanism and a selective predisposition to mycobacterial disease.
Our finding is unexpected because MCTS1 is ubiquitously expressed and has a fundamental molecular function in the dissociation of deacylated tRNAs from post-termination 40S ribosomal complexes during ribosome recycling (on mORF stop codons) and re-initiation (on uORF stop codons). The translation re-initiation functions of DENR and MCTS1 are essentially identical in amplitude 58,59,62. However, DENRKO is embryonically lethal in mice, whereas MCTS1KO mice are viable 60. This is consistent with the more severe ribosome recycling defect of DENRKO HeLa cells than of MCTS1KO HeLa cells. Furthermore, we found no MSMD patient homozygous for rare candidate DENR variants. The apparent lack or extreme rarity of DENR-deficient patients suggests that this deficiency may be embryonically lethal in humans, as it is in mice. This contrasts with our findings that all five patients with MCTS1 deficiency have isolated MSMD and are otherwise healthy at ages of six months to 18 years. However, only one MCTS1-deficient patient has yet reached adulthood, and other clinical phenotypes may appear as the patients age.
We define the set of human MCTS1-dependent genes in cells from MCTS1-deficient patients. We identify JAK2 as a translational target of DENR-MCTS1 and probably the key contributor to MSMD. Decreases in the levels of other MCTS1-dependent proteins do not appear to have clinical consequences. Surprisingly, partial JAK2 deficiency impairs cellular responses to IL-23 and the subsequent induction of IFN-γ in lymphocytes, but not other cytokine response pathways involving JAK2. DENRKO has been reported to impair JAK2 translation and subsequent responses to IFN- in mouse tumor cells85. Responses to IL-12 and IL-23 were not tested in this previous study. We found the same connection between MCTS1 and JAK2 in human cells, but the response to IFN- was intact. Low levels of JAK2 may have a much greater effect in IL-23- and IL-12-responsive cells than in IFN--responsive cells. It is probably no coincidence that the four types of MSMD-causing TYK2 variants, including the common P1104A variant, have an impact on the IL-23 response pathway, whereas only two of these types of variants also impair responses to IL-1235. This finding suggests that the IL-23R is more dependent on qualitative and quantitative variations of JAK2 or TYK2 than the IL-12R. Biochemical differences may be responsible for the weaker IL-23R signaling observed.
Our findings support our previous reports that human IL-23 is a key IFN--inducing cytokine18,30,42,73. Despite having the p40 chain in common, IL-23 has historically been seen as an IL-17-inducing cytokine73,88-91, whereas IL-12 is seen as the key IFN--inducing cytokine73,91. Recent discoveries have suggested that human IL-23 plays a key role in IFN--mediated immunity to mycobacteria, and a more minor role in IL-17-mediated immunity to fungi. First, AR complete IL-23R deficiency underlies MSMD with almost complete clinical penetrance but only a mild form of CMC with incomplete penetrance30,42. Second, homozygosity for the common TYK2 P1104A allele selectively impairs responses to IL-23 and underlies MSMD with low penetrance and tuberculosis with high penetrance, but has no effect on susceptibility to CMC18,54. Third, impairment of the cellular response to IL-23 is the only mechanism underlying mycobacterial disease common to patients with the five forms of AR TYK2 deficiency28,60. Fourth, MCTS1-deficient patients have impaired cellular responses to IL-23 and isolated MSMD, with no detectable IL-17 deficiency or CMC. Collectively, these observations suggest that human IL-23 acts more as an IFN-inducing antimycobacterial cytokine than as an IL-17-inducing antifungal cytokine.
We found that IFN- induction in response to IL-23 stimulation was impaired in the V2+ T and MAIT cells of MCTS1-deficient patients. This finding is consistent with previous reports, as V2+ T and MAIT cells are among the subsets producing the largest amounts of IFN- in response to IL-2330,42. Furthermore, the IFN- production of these cells in response to IL-23 is impaired in IL-12R1-/-, IL-23R-/- and TYK2-/- individuals30,42,60. NK cells are also potent inducers of IFN- upon stimulation with IL-23, but their transcriptional response to IL-23 is only moderately impaired in MCTS1-deficient patients. Interestingly, the induction of IFN- was also impaired in the patients’ TH1* cells. TH1* cells have a lower level of IFN- induction than MAIT, V2+ T and NK cells. Nonetheless, TH1* cells display more than an eight-fold induction of IFN- in response to IL-23 (Figure S8F), this induction being totally abolished in MCTS1-, IL-12R1-, and IL-23R-deficient patients. TH1* cells are the major lymphocyte subset producing IFN- after exposure to BCG in vitro92,93. The counts of these cells in the blood increase in individuals with latent tuberculosis infection94. IL-23 may, thus, also contribute to TH1*-mediated immunity to mycobacteria.
Finally, we can speculate about the clinical phenotype of MCTS1-deficient patients in the absence of BCG vaccination. Surprisingly, none of the six MCTS1-deficient individuals had EM disease. This may be due to the protection afforded by BCG disease, as seen in IL-12Rß1-deficient patients38,39. It may also reflect an incomplete IFN- deficit, as only one of the seven reported IL-23R-deficient patients suffered from EM disease41,42 and cellular responses to IL-23 are impaired, but not abolished in MCTS1-deficient patients. In regions of endemic tuberculosis, in which BCG vaccination is not widely or efficiently performed, or if penetrance for BCG disease is incomplete, MCTS1 deficiency may be revealed by severe tuberculosis. Overall, this study reveals a surprising mechanistic connection between deficiency of a basic, ubiquitous, biochemical mechanism and a selective predisposition to mycobacterial disease.