A Novel STK4 Mutation Impairs T Cell Immunity Through Dysregulation of Cytokine-Induced Adhesion and Chemotaxis Genes.

PURPOSE: Human serine/threonine kinase 4 (STK4) deficiency is a rare, autosomal recessive genetic disorder leading to combined immunodeficiency; however, the extent to which immune signaling and host defense are impaired is unclear. We assessed the functional consequences of a novel, homozygous nonsense STK4 mutation (NM_006282.2:c.871C > T, p.Arg291*) identified in a pediatric patient by comparing his innate and adaptive cell-mediated and humoral immune responses with those of three heterozygous relatives and unrelated controls. METHODS: The genetic etiology was verified by whole genome and Sanger sequencing. STK4 gene and protein expression was measured by quantitative RT-PCR and immunoblotting, respectively. Cellular abnormalities were assessed by high-throughput RT-RCR, RNA-Seq, ELISA, and flow cytometry. Antibody responses were assessed by ELISA and phage immunoprecipitation-sequencing. RESULTS: The patient exhibited partial loss of STK4 expression and complete loss of STK4 function combined with recurrent viral and bacterial infections, notably persistent Epstein-Barr virus viremia and pulmonary tuberculosis. Cellular and molecular analyses revealed abnormal fractions of T cell subsets, plasmacytoid dendritic cells, and NK cells. The transcriptional responses of the patient's whole blood and PBMC samples indicated dysregulated interferon signaling, impaired T cell immunity, and increased T cell apoptosis as well as impaired regulation of cytokine-induced adhesion and leukocyte chemotaxis genes. Nonetheless, the patient had detectable vaccine-specific antibodies and IgG responses to various pathogens, consistent with a normal CD19 + B cell fraction, albeit with a distinctive antibody repertoire, largely driven by herpes virus antigens. CONCLUSION: Patients with STK4 deficiency can exhibit broad impairment of immune function extending beyond lymphoid cells.

Inherited human IRAK-1 deficiency selectively impairs TLR signaling in fibroblasts.

Most members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) families transduce signals via a canonical pathway involving the MyD88 adapter and the interleukin-1 receptor-associated kinase (IRAK) complex. This complex contains four molecules, including at least two (IRAK-1 and IRAK-4) active kinases. In mice and humans, deficiencies of IRAK-4 or MyD88 abolish most TLR (except for TLR3 and some TLR4) and IL-1R signaling in both leukocytes and fibroblasts. TLR and IL-1R responses are weak but not abolished in mice lacking IRAK-1, whereas the role of IRAK-1 in humans remains unclear. We describe here a boy with X-linked MECP2 deficiency-related syndrome due to a large de novo Xq28 chromosomal deletion encompassing both MECP2 and IRAK1 Like many boys with MECP2 null mutations, this child died very early, at the age of 7 mo. Unlike most IRAK-4- or MyD88-deficient patients, he did not suffer from invasive bacterial diseases during his short life. The IRAK-1 protein was completely absent from the patient's fibroblasts, which responded very poorly to all TLR2/6 (PAM2CSK4, LTA, FSL-1), TLR1/2 (PAM3CSK4), and TLR4 (LPS, MPLA) agonists tested but had almost unimpaired responses to IL-1ß. By contrast, the patient's peripheral blood mononuclear cells responded normally to all TLR1/2, TLR2/6, TLR4, TLR7, and TLR8 (R848) agonists tested, and to IL-1ß. The death of this child precluded long-term evaluations of the clinical consequences of inherited IRAK-1 deficiency. However, these findings suggest that human IRAK-1 is essential downstream from TLRs but not IL-1Rs in fibroblasts, whereas it plays a redundant role downstream from both TLRs and IL-1Rs in leukocytes.

Staphylococcal-associated molecular patterns enhance expression of immune defense genes induced by IL-17 in mammary epithelial cells.

Interleukin-17A (IL-17A) and IL-17F have been shown to mediate a crucial crosstalk between the immune system and various epithelial tissues, stimulating various defensive mechanisms to bacterial infections. A number of studies have characterized the response to IL-17A and IL-17F of epithelial cells from airways, intestine, and skin, but not from the mammary gland. To evaluate the potential contribution of IL-17 to the immune defense of the mammary gland, we analyzed the effects of recombinant bovine IL-17A and IL-17F on primary bovine mammary epithelial cells (MEC) by quantitative PCR and ELISA. We found expression (mRNA) of the two components of the IL-17 receptor complex, IL-17RA and IL-17RC, in mammary tissue and MEC in vitro. The expression of a number of genes encoding cytokines, chemokines and proteins endowed with antibacterial activities was increased by IL-17A, and to a lesser extent by IL-17F, but the magnitude of responses was modest. As expected, responses were augmented by the combination of IL-17A or IL-17F with TNF-α. Interestingly, responses of a few of the tested genes, such as IL8, CCL20, iNOS, and CfB, were augmented by the combination of IL-17A with staphylococcal lipoteichoic acid or muramyl dipeptide, bacterial agonists of the innate immune system. This can be interpreted as indicating that IL-17A and IL-17F are tailored to exert their full potential in a septic environment. MEC responses were characterized by the expression of chemokines targeting not only neutrophils (CXCL3 and CXCL8) but also mononuclear leucocytes (CCL2, CCL20). Production of IL-6 was low and the inflammatory cytokines TNF-α and IL-1ß were expressed (mRNA) but proteins were not secreted. Altogether, our results suggest that IL-17A and IL-17F have a potential to modulate the mammary gland immune response to mastitis-causing pathogens.

A curated transcriptome dataset collection to investigate inborn errors of immunity.

Primary immunodeficiencies (PIDs) are a heterogeneous group of inherited disorders, frequently caused by loss-of-function and less commonly by gain-of-function mutations, which can result in susceptibility to a broad or a very narrow range of infections but also in inflammatory, allergic or malignant diseases. Owing to the wide range in clinical manifestations and variability in penetrance and expressivity, there is an urgent need to better understand the underlying molecular, cellular and immunological phenotypes in PID patients in order to improve clinical diagnosis and management. Here we have compiled a manually curated collection of public transcriptome datasets mainly obtained from human whole blood, peripheral blood mononuclear cells (PBMCs) or fibroblasts of patients with PIDs and of control subjects for subsequent meta-analysis, query and interpretation. A total of nineteen (19) datasets derived from studies of PID patients were identified and retrieved from the NCBI Gene Expression Omnibus (GEO) database and loaded in GXB, a custom web application designed for interactive query and visualization of integrated large-scale data. The dataset collection includes samples from well characterized PID patients that were stimulated ex vivo under a variety of conditions to assess the molecular consequences of the underlying, naturally occurring gene defects on a genome-wide scale. Multiple sample groupings and rank lists were generated to facilitate comparisons of the transcriptional responses between different PID patients and control subjects. The GXB tool enables browsing of a single transcript across studies, thereby providing new perspectives on the role of a given molecule across biological systems and PID patients. This dataset collection is available at http://pid.gxbsidra.org/dm3/geneBrowser/list.

A curated transcriptome dataset collection to investigate the blood transcriptional response to viral respiratory tract infection and vaccination.

The human immune defense mechanisms and factors associated with good versus poor health outcomes following viral respiratory tract infections (VRTI), as well as correlates of protection following vaccination against respiratory viruses, remain incompletely understood. To shed further light into these mechanisms, a number of systems-scale studies have been conducted to measure transcriptional changes in blood leukocytes of either naturally or experimentally infected individuals, or in individual's post-vaccination. Here we are making available a public repository, for research investigators for interpretation, a collection of transcriptome datasets obtained from human whole blood and peripheral blood mononuclear cells (PBMC) to investigate the transcriptional responses following viral respiratory tract infection or vaccination against respiratory viruses. In total, Thirty one31 datasets, associated to viral respiratory tract infections and their related vaccination studies, were identified and retrieved from the NCBI Gene Expression Omnibus (GEO) and loaded in a custom web application designed for interactive query and visualization of integrated large-scale data. Quality control checks, using relevant biological markers, were performed. Multiple sample groupings and rank lists were created to facilitate dataset query and interpretation. Via this interface, users can generate web links to customized graphical views, which may be subsequently inserted into manuscripts to report novel findings. The GXB tool enables browsing of a single gene across projects, providing new perspectives on the role of a given molecule across biological systems in the diagnostic and prognostic following VRTI but also in identifying new correlates of protection. This dataset collection is available at: http://vri1.gxbsidra.org/dm3/geneBrowser/list.

Chronic mucocutaneous candidiasis and connective tissue disorder in humans with impaired JNK1-dependent responses to IL-17A/F and TGF-ß.

Genetic etiologies of chronic mucocutaneous candidiasis (CMC) disrupt human IL-17A/F-dependent immunity at mucosal surfaces, whereas those of connective tissue disorders (CTDs) often impair the TGF-ß-dependent homeostasis of connective tissues. The signaling pathways involved are incompletely understood. We report a three-generation family with an autosomal dominant (AD) combination of CMC and a previously undescribed form of CTD that clinically overlaps with Ehlers-Danlos syndrome (EDS). The patients are heterozygous for a private splice-site variant of MAPK8, the gene encoding c-Jun N-terminal kinase 1 (JNK1), a component of the MAPK signaling pathway. This variant is loss-of-expression and loss-of-function in the patients' fibroblasts, which display AD JNK1 deficiency by haploinsufficiency. These cells have impaired, but not abolished, responses to IL-17A and IL-17F. Moreover, the development of the patients' TH17 cells was impaired ex vivo and in vitro, probably due to the involvement of JNK1 in the TGF-ß-responsive pathway and further accounting for the patients' CMC. Consistently, the patients' fibroblasts displayed impaired JNK1- and c-Jun/ATF-2-dependent induction of key extracellular matrix (ECM) components and regulators, but not of EDS-causing gene products, in response to TGF-ß. Furthermore, they displayed a transcriptional pattern in response to TGF-ß different from that of fibroblasts from patients with Loeys-Dietz syndrome caused by mutations of TGFBR2 or SMAD3, further accounting for the patients' complex and unusual CTD phenotype. This experiment of nature indicates that the integrity of the human JNK1-dependent MAPK signaling pathway is essential for IL-17A- and IL-17F-dependent mucocutaneous immunity to Candida and for the TGF-ß-dependent homeostasis of connective tissues.

IRF4 haploinsufficiency in a family with Whipple's disease.

Most humans are exposed to Tropheryma whipplei (Tw). Whipple's disease (WD) strikes only a small minority of individuals infected with Tw (<0.01%), whereas asymptomatic chronic carriage is more common (<25%). We studied a multiplex kindred, containing four WD patients and five healthy Tw chronic carriers. We hypothesized that WD displays autosomal dominant (AD) inheritance, with age-dependent incomplete penetrance. We identified a single very rare non-synonymous mutation in the four patients: the private R98W variant of IRF4, a transcription factor involved in immunity. The five Tw carriers were younger, and also heterozygous for R98W. We found that R98W was loss-of-function, modified the transcriptome of heterozygous leukocytes following Tw stimulation, and was not dominant-negative. We also found that only six of the other 153 known non-synonymous IRF4 variants were loss-of-function. Finally, we found that IRF4 had evolved under purifying selection. AD IRF4 deficiency can underlie WD by haploinsufficiency, with age-dependent incomplete penetrance.

T helper 17-associated cytokines are produced during antigen-specific inflammation in the mammary gland.

Infectious mastitis cuts down milk production profitability and is a major animal welfare problem. Bacteria-induced inflammation in the mammary gland (MG) is driven by innate immunity, but adaptive immunity can modulate the innate response. Several studies have shown that it is possible to elicit inflammation in the MG by sensitization to an antigen subsequently infused into the lumen of the gland. The objective of our study was to characterize the inflammation triggered in the MG of cows sensitized to ovalbumin, by identifying the cytokines and chemokines likely to play a part in the reaction. Among immunized cows, responders mobilized locally high numbers of leukocytes. An overexpression of the genes encoding IL-17a, IL-17F, IL-21, IL-22 and INF-γ was found in milk cell RNA extracts in the early phase of the inflammatory response. At the protein level, IL-17A was detected in milk as soon as the first sampling time (8 h post-challenge), and both IL-17A and IFN-γ concentrations peaked at 12 to 24 h post-challenge. In mammary tissue from challenged quarters, overexpression of the genes encoding IL-17A, IL-17F, IL-21, IL-22, IL-26 and IFN-γ was observed. Neutrophil-attracting chemokines (CXCL3 and CXCL8) were found in milk, and overexpressed transcripts of chemokines attracting lymphocytes and other mononuclear leukocytes (CXCL10, CCL2, CCL5, CCL20) were detected in mammary tissue. Expression of IL-17A, as revealed by immunohistochemistry, was located in epithelial cells, in leukocytes in the connective tissue and in association with the epithelium, and in migrated alveolar leukocytes of challenged quarters. Altogether, these results show that antigen-specific inflammation in the MG was characterized by the production of IL-17 and IFN-γ. The orientation of the inflammatory response induced by the antigen-specific response has the potential to strongly impact the outcome of bacterial infections of the MG.

Molecular interaction and functional coupling between type 3 inositol 1,4,5-trisphosphate receptor and BKCa channel stimulate breast cancer cell proliferation.

BACKGROUND: The implication of ion channels and inositol 1,4,5-trisphosphate (IP3)-induced Ca(2+) signalling (IICS) in the carcinogenesis processes, including deregulation of cell proliferation, migration and invasion, is increasingly studied. Studies from our laboratory have shown that type 3 IP3 receptor (IP3R3) and voltage- and Ca(2+)-dependent K(+) channels BKCa channels are involved in human breast cancer cell proliferation. In this context, we investigated the probable interaction between these two proteins (IP3R3 and BKCa channel) in normal and in breast cancer cells. METHODS: MCF-7 and MCF-10A cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay in the presence or absence of adenosine triphosphate (ATP). Furthermore, cell-cycle analysis was carried out and cell cycle protein expression was examined by Western blotting. Immunocytochemistry and co-immunoprecipitation assays were used to check co-localisation of BKCa and IP3R3 and their molecular interaction. Finally, whole cell patch-clamp and Ca(2+) imaging were performed to assess the functional interaction. RESULTS: Our results are in favour of a functional and a molecular coupling between IP3R3 and BKCa channel that is involved in MCF-7 proliferation. Indeed, ATP increased MCF-7 cell proliferation and this effect was impaired when the expression of BKCa and/or IP3R3 has been reduced by specific small interfering RNAs (siRNAs). Flow cytometry experiments showed that both siRNAs led to cell cycle arrest in the G0/G1 phase and these results were confirmed by the analysis of cell cycle protein expression. Specifically, BKCa and IP3R3 silencing decreased both cyclin-D1 and cyclin-dependent kinase 4 (CDK4) expression levels. Furthermore, ATP elicited a phospholipase C (PLC)-dependent elevation of internal Ca(2+) that triggered in turn an iberiotoxin (IbTx)- and a tetra-ethyl-ammonium (TEA)-sensitive membrane hyperpolarisation that was strongly reduced in the cells with silenced IP3R3 or BKCa. In the same way, intracellular application of Ins(2,4,5)P3 triggered an IbTx-sensitive membrane hyperpolarisation. Moreover, intracellular Ca(2+) chelation with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) prevented ATP-induced BKCa activation. BKCa and IP3R3 also co-immunoprecipitated and this interaction seemed to occur in cholesterol-enriched microdomains. Conversely, in the normal breast cell line MCF-10A, neither ATP application nor BKCa silencing affected cell proliferation. Furthermore, IP3R3 and BKCa did not co-immunoprecipitate, suggesting the absence of a molecular coupling between BKCa and IP3R3 in the MCF-10A normal cell line. CONCLUSION: Altogether, our results suggest a molecular and functional link between BKCa channel and IP3R3 in cancer cells. Our findings led us to propose this coupling between BKCa and IP3R3 as an important mechanism for tumour cell proliferation.

Muramyl dipeptide synergizes with Staphylococcus aureus lipoteichoic acid to recruit neutrophils in the mammary gland and to stimulate mammary epithelial cells.

Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1ß were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.