Patients with STAT1 Gain-of-function Mutations Display Increased Apoptosis which is Reversed by the JAK Inhibitor Ruxolitinib.

INTRODUCTION: The signal transducer and activator of transcription (STAT1) gain-of-function (GOF) syndrome accounts for most cases of chronic mucocutaneous candidiasis but is characterized by a broader clinical phenotype that may include bacterial, viral, or invasive fungal infections, autoimmunity, autoinflammatory manifestations, vascular complications, or malignancies. The severity of lymphopenia may vary and influence the infectious morbidity. METHODS: In our cohort of seven STAT1-GOF patients, we investigated the mechanisms that may determine T lymphopenia, we characterized the interferon gene signature (IGS) and analyzed the effect of ruxolitinib in reverting the immune dysregulation. RESULTS: STAT1-GOF patients exhibited increased T lymphocyte apoptosis that was significantly augmented in both resting conditions and following stimulation with mitogens and IFNα, as evaluated by flow cytometry by Annexin V/ Propidium iodide assay. The JAK inhibitor ruxolitinib significantly reduced the IFNα-induced hyperphosphorylation of STAT1 and reverted the stimulation-induced T-cell apoptosis, in vitro. In two adult STAT1-GOF patients, the JAKinib treatment ameliorated chronic mucocutaneous candidiasis and lymphopenia. Most STAT1-GOF patients, particularly those who had autoimmunity, presented increased IGS that significantly decreased in the two patients during ruxolitinib treatment. CONCLUSION: In STAT1-GOF patients, T lymphocyte apoptosis is increased, and T lymphopenia may determine higher risk of severe infections. The JAKinib target therapy should be evaluated to treat severe chronic candidiasis and lymphopenia, and to downregulate the IFNs in patients with autoinflammatory or autoimmune manifestations.

Immuno-Contexture and Immune Checkpoint Molecule Expression in Mismatch Repair Proficient Colorectal Carcinoma.

Colorectal carcinoma (CRC) represents a lethal disease with heterogeneous outcomes. Only patients with mismatch repair (MMR) deficient CRC showing microsatellite instability and hyper-mutated tumors can obtain clinical benefits from current immune checkpoint blockades; on the other hand, immune- or target-based therapeutic strategies are very limited for subjects with mismatch repair proficient CRC (CRCpMMR). Here, we report a comprehensive typing of immune infiltrating cells in CRCpMMR. We also tested the expression and interferon-γ-modulation of PD-L1/CD274. Relevant findings were subsequently validated by immunohistochemistry on fixed materials. CRCpMMR contain a significantly increased fraction of CD163+ macrophages (TAMs) expressing TREM2 and CD66+ neutrophils (TANs) together with decrease in CD4-CD8-CD3+ double negative T lymphocytes (DNTs); no differences were revealed by the analysis of conventional and plasmacytoid dendritic cell populations. A fraction of tumor-infiltrating T-cells displays an exhausted phenotype, co-expressing PD-1 and TIM-3. Remarkably, expression of PD-L1 on fresh tumor cells and TAMs was undetectable even after in vitro stimulation with interferon-γ. These findings confirm the immune suppressive microenvironment of CRCpMMR characterized by dense infiltration of TAMs, occurrence of TANs, lack of DNTs, T-cell exhaustion, and interferon-γ unresponsiveness by host and tumor cells. Appropriate bypass strategies should consider these combinations of immune escape mechanisms in CRCpMMR.

Circulation of Respiratory Viruses in Hospitalized Adults before and during the COVID-19 Pandemic in Brescia, Italy: A Retrospective Study.

Different preventive public health measures were adopted globally to limit the spread of SARS-CoV-2, such as hand hygiene and the use of masks, travel restrictions, social distance actions such as the closure of schools and workplaces, case and contact tracing, quarantine and lockdown. These measures, in particular physical distancing and the use of masks, might have contributed to containing the spread of other respiratory viruses that occurs principally by contact and droplet routes. The aim of this study was to evaluate the prevalence of different respiratory viruses (influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, 3 and 4, rhinovirus, adenovirus, metapneumovirus and human coronaviruses) after one year of the pandemic. Furthermore, another aim was to evaluate the possible impact of these non-pharmaceutical measures on the circulation of seasonal respiratory viruses. This single center study was conducted between January 2017-February 2020 (pre-pandemic period) and March 2020-May 2021 (pandemic period). All adults >18 years with respiratory symptoms and tested for respiratory pathogens were included in the study. Nucleic acid detection of all respiratory viruses was performed by multiplex real time PCR. Our results show that the test positivity for influenza A and B, metapneumovirus, parainfluenza virus, respiratory syncytial virus and human coronaviruses decreased with statistical significance during the pandemic. Contrary to this, for adenovirus the decrease was not statistically significant. Conversely, a statistically significant increase was detected for rhinovirus. Coinfections between different respiratory viruses were observed during the pre-pandemic period, while the only coinfection detected during pandemic was between SARS-CoV-2 and rhinovirus. To understand how the preventive strategies against SARS-CoV-2 might alter the transmission dynamics and epidemic patterns of respiratory viruses is fundamental to guide future preventive recommendations.

Case Report: Analysis of Inflammatory Cytokines IL-6, CCL2/MCP1, CCL5/RANTES, CXCL9/MIG, and CXCL10/IP10 in a Cystic Fibrosis Patient Cohort During the First Wave of the COVID-19 Pandemic.

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, data registered in the European countries revealed increasing cases of infection in cystic fibrosis (CF) patients. In the course of this pandemic, we enrolled 17 CF patients for a study evaluating inflammatory markers. One of them developed COVID-19, giving us the possibility to analyze inflammatory markers in the acute phase as compared to levels detected before and after the infectious episode and to levels measured in the other CF patients enrolled to the study who did not experience COVID-19 and 23 patients referred to our center for SARS-CoV-2 infection.

Case Report: Hypomorphic Function and Somatic Reversion in DOCK8 Deficiency in One Patient With Two Novel Variants and Sclerosing Cholangitis.

DOCK8 deficiency is a combined immunodeficiency due to biallelic variants in dedicator of cytokinesis 8 (DOCK8) gene. The disease has a wide clinical spectrum encompassing recurrent infections (candidiasis, viral and bacterial infections), virally driven malignancies and immune dysregulatory features, including autoimmune (cytopenia and vasculitis) as well as allergic disorders (eczema, asthma, and food allergy). Hypomorphic function and somatic reversion of DOCK8 has been reported to result in incomplete phenotype without IgE overproduction. Here we describe a case of DOCK8 deficiency in a 8-year-old Caucasian girl. The patient's disease was initially classified as autoimmune thrombocytopenia, which then evolved toward a combined immunodeficiency phenotype with recurrent infections, persistent EBV infection and lymphoproliferation. Two novel variants (one deletion and one premature stop codon) were characterized, resulting in markedly reduced, but not absent, DOCK8 expression. Somatic reversion of the DOCK8 deletion was identified in T cells. Hypomorphic function and somatic reversion were associated with restricted T cell repertoire, decreased STAT5 phosphorylation and impaired immune synapse functioning in T cells. Although the patient presented with incomplete phenotype (absence of markedly increase IgE and eosinophil count), sclerosing cholangitis was incidentally detected, thus indicating that hypomorphic function and somatic reversion of DOCK8 may delay disease progression but do not necessarily prevent from severe complications.

Transient Decrease of Circulating and Tissular Dendritic Cells in Patients With Mycobacterial Disease and With Partial Dominant IFNγR1 Deficiency.

Interferon-γ receptor 1 (IFNγR1) deficiency is one of the inborn errors of IFN-γ immunity underlying Mendelian Susceptibility to Mycobacterial Disease (MSMD). This molecular circuit plays a crucial role in regulating the interaction between dendritic cells (DCs) and T lymphocytes, thus affecting DCs activation, maturation, and priming of T cells involved in the immune response against intracellular pathogens. We studied a girl who developed at the age of 2.5 years a Mycobacterium avium infection characterized by disseminated necrotizing granulomatous lymphadenitis, and we compared her findings with other patients with the same genetic condition. The patient carried a heterozygous 818del4 mutation in the IFNGR1 gene responsible of autosomal dominant (AD) partial IFNγR1 deficiency. During the acute infection blood cells immunophenotyping showed a marked reduction in DCs counts, including both myeloid (mDCs) and plasmacytoid (pDCs) subsets, that reversed after successful prolonged antimicrobial therapy. Histology of her abdomen lymph node revealed a profound depletion of tissue pDCs, as compared to other age-matched granulomatous lymphadenitis of mycobacterial origin. Circulating DCs depletion was also observed in another patient with AD partial IFNγR1 deficiency during mycobacterial infection. To conclude, AD partial IFNγR1 deficiency can be associated with a transient decrease in both circulating and tissular DCs during acute mycobacterial infection, suggesting that DCs counts monitoring might constitute a useful marker of treatment response.

STAT mutations as program switchers: turning primary immunodeficiencies into autoimmune diseases.

STAT proteins are a family of transcription factors that mediate cellular response to cytokines and growth factors. Study of patients with familial susceptibility to pathogens and/or autoimmune diseases has led to the identification of 7 inherited disorders that are caused by mutations of 4 STAT family genes. Homozygous or compound heterozygous mutations of STAT1 lead to complete or partial forms of STAT1 deficiency that are associated with susceptibility to intracellular pathogens and herpetic infections. Patients with heterozygous STAT1 gain-of-function (GOF) mutations usually present with chronic mucocutaneous candidiasis (CMC) but may also experience bacterial and viral infections, autoimmune manifestations, lymphopenia, cerebral aneurysms, and increased risk to develop tumors. STAT2 deficiency has been described in 5 family members and is characterized by selective susceptibility to viral infections, whereas STAT3 loss-of-function (LOF) mutations are causative of the autosomal-dominant hyper-IgE syndrome (HIES), a condition that is characterized by cutaneous and respiratory infections in association with mucocutaneous candidiasis, eczema, skeletal and connective tissue abnormalities, eosinophilia, and high levels IgE. STAT5B LOF and STAT3 GOF mutations are both associated with disorders characterized by autoimmune or allergic manifestations, together with increased risk of infections. Particularly, STAT5b deficiency results in growth hormone (GH) insensitivity, immunodeficiency, diarrhea, and generalized eczema, whereas STAT3 GOF mutations result in autoimmune cytopenia, lymphadenopathy, short stature, infections, enteropathy, and multiorgan autoimmunity, including early-onset type I diabetes, thyroiditis, hepatitis, arthritis, and interstitial lung disease.

SH2-domain mutations in STAT3 in hyper-IgE syndrome patients result in impairment of IL-10 function.

Autosomal-dominant hyper-IgE syndrome (AD-HIES) is a primary immunodeficiency caused by STAT3 mutations. This inherited condition is characterized by eczema, staphylococcal cold abscesses and recurrent pulmonary infections. Given that STAT3 is involved in IL-10 signaling, we examined the immunoregulatory role of IL-10 in inflammation by studying the effects of IL-10 on monocytes, neutrophils and monocyte-derived DCs from HIES subjects. Analysis of gene expression in PBMCs and neutrophils isolated from HIES patients and stimulated with LPS in the presence of IL-10 showed reduced expression of IL1RN, which encodes IL-1 receptor antagonist (IL-1ra), and SOCS3 mRNA but increased CXCL8 mRNA expression. Moreover, secretion of the anti-inflammatory protein IL-1ra was reduced in AD-HIES patients. DCs from HIES patients secreted higher levels of TNF-α, IL-6 and, to a lesser extent, IL-12 when these cells were cultured in the presence of IL-10. These results suggest that IL-10 activity is affected in myeloid cells (e.g. monocytes, DCs) of HIES patients. Impairment of IL-10 signaling in patients with AD-HIES might result in an altered balance between pro-inflammatory and anti-inflammatory signals and might lead to persistent inflammation and delayed healing after infections.

Uncovering an IL-10-dependent NF-kappaB recruitment to the IL-1ra promoter that is impaired in STAT3 functionally defective patients.

The interleukin 1 receptor antagonist (IL-1ra) is an important negative regulator of the inflammatory response, whose genetic deficiency has been recently shown to cause a severe autoinflammatory syndrome in humans. In this study we characterized the molecular mechanisms whereby interleukin 10 (IL-10) potentiates IL-1ra transcription in LPS-stimulated monocytes and neutrophils. Using chromatin immunoprecipitation, we show that although NF-kappaBp65 and NF-kappaBp50 proteins accumulate into the nuclei and bind to the IkappaB alpha promoter during LPS stimulation, they are not recruited to the kappaB sites of the IL-1ra promoter. However, in response to LPS plus IL-10, which were found to induce chromatin acetylation, recruitment of both NF-kappaBp65 and NF-kappaBp50 to the IL-1ra promoter efficiently occurs in a STAT3-dependent manner. Accordingly, in neutrophils from hyper-IgE syndrome patients, who carry a nonfunctional STAT3, IL-10 failed to promote NF-kappaBp65 recruitment to the IL-1ra promoter and consequently to potentiate LPS-induced IL-1ra transcription. Altogether our findings uncover a novel mechanism whereby IL-10-activated STAT3 modulates IL-1ra transcription in LPS-treated phagocytes by making IL-1ra promoter accessible to readily available nuclear NF-kappaB.