Inherited human c-Rel deficiency disrupts myeloid and lymphoid immunity to multiple infectious agents.

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient's naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.

Response to Oxidative Burst-Induced Hypoxia Is Associated With Macrophage Inflammatory Profiles as Revealed by Cellular Genome-Wide Association.

Background: In mammalian species, hypoxia is a prominent feature of inflammation. The role of hypoxia in regulating macrophage responses via alteration in metabolic pathways is well established. Recently, oxidative burst-induced hypoxia has been shown in murine macrophages after phagocytosis. Despite the available detailed information on the regulation of macrophage function at transcriptomic and epigenomic levels, the association of genetic polymorphism and macrophage function has been less explored. Previously, we have shown that host genetics controls approximately 80% of the variation in an oxidative burst as measured by nitric oxide (NO-). Further studies revealed two clusters of transcription factors (hypoxia-related and inflammatory-related) are under the genetic control that shapes macrophages' pro-inflammatory characteristics. Material and Methods: In the current study, the association between 43,066 autosomal Single Nucleic Polymorphism (SNPs) and the ability of MDMs in production of NO- in response to E. coli was evaluated in 58 Holstein cows. The positional candidate genes near significant SNPs were selected to perform functional analysis. In addition, the interaction between the positional candidate genes and differentially expressed genes from our previous study was investigated. Results: Sixty SNPs on 22 chromosomes of the bovine genome were found to be significantly associated with NO- production of macrophages. The functional genomic analysis showed a significant interaction between positional candidate genes and mitochondria-related differentially expressed genes from the previous study. Further examination showed 7 SNPs located in the vicinity of genes with roles in response to hypoxia, shaping approximately 73% of the observed individual variation in NO- production by MDM. Regarding the normoxic condition of macrophage culture in this study, it was hypothesized that oxidative burst is responsible for causing hypoxia at the cellular level. Conclusion: The results suggest that the genetic polymorphism via regulation of response to hypoxia is a candidate step that perhaps shapes macrophage functional characteristics in the pathway of phagocytosis leading to oxidative burst, hypoxia, cellular response to hypoxia and finally the pro-inflammatory responses. Since all cells in one individual carry the same alleles, the effect of genetic predisposition of sensitivity to hypoxia will likely be notable on the clinical outcome to a broad range of host-pathogen interactions.

Transcriptomic Profiles of Monocyte-Derived Macrophages in Response to Escherichia coli is Associated with the Host Genetics.

Reactive Nitrogen Species (RNS) are a group of bactericidal molecules produced by macrophages in response to pathogens in a process called oxidative burst. Nitric oxide (NO-) is a member of RNS produced from arginine by inducible Nitric Oxide Synthase (iNOS) enzyme. The activity of iNOS and production of NO- by macrophages following stimulation is one of the indicators of macrophage polarization towards M1/proinflammatory. Production of NO- by bovine monocyte-derived macrophage (MDM) and mouse peritoneal macrophages has been shown to be strongly associated with host genetic with the heritability of 0.776 in bovine MDM and 0.8 in mouse peritoneal macrophages. However, the mechanism of genetic regulation of macrophage response has remained less explored. In the current study, the transcriptome of bovine MDMs was compared between two extreme phenotypes that had been classified as high and low responder based on NO- production. The results showed that 179 and 392 genes were differentially expressed (DE) between high and low responder groups at 3 and 18 hours after exposure to Escherichia coli, respectively. A set of 11 Transcription Factors (TFs) (STAT1, IRF7, SPI1, STAT4, IRF1, HIF1A, FOXO3, REL, NFAT5, HIC1, and IRF4) at 3 hours and a set of 13 TFs (STAT1, IRF1, HIF1A, STAT4, ATF4, TP63, EGR1, CDKN2A, RBL1, E2F1, PRDM1, GATA3, and IRF4) at 18 hours after exposure to E. coli were identified to be differentially regulated between the high and low responder phenotypes. These TFs were found to be divided into two clusters of inflammatory- and hypoxia-related TFs. Functional analysis revealed that some key canonical pathways such as phagocytosis, chemotaxis, antigen presentation, and cell-to-cell signalling are enriched among the over-expressed genes by high responder phenotype. Based on the results of this study, it was inferred that the functional characteristics of bovine MDMs are associated with NO-based classification. Since NO- production is strongly associated with host genetics, this study for the first time shows the distinct proinflammatory profiles of macrophages are controlled by the natural genetic polymorphism in an outbred population. In addition, the results suggest that genetics can be considered as a new dimension in the current model of macrophage polarization which is currently described by the combination of stimulants, only.

The effect of host genetics on in vitro performance of bovine monocyte-derived macrophages.

The dynamic interaction between the host and pathogens, along with environmental factors, influences the regulation of mammalian immune responses. Therefore, comprehensive in vivo immune-phenotyping during an active response to a pathogen can be complex and prone to confounding effects. Evaluating critical fundamental aspects of the immune system at a cellular level is an alternative approach to reduce this complexity. Therefore, the objective of the current study was to examine an in vitro model for functional phenotyping of bovine monocyte-derived macrophages (MDM), cells which play a crucial role at all phases of inflammation, as well influence downstream immune responses. As indicators of MDM function, phagocytosis and nitric oxide (NO-) production were tested in MDM of 16 cows in response to 2 common bacterial pathogens of dairy cows, Escherichia coli and Staphylococcus aureus. Notable functional variations were observed among the individuals (coefficient of variation: 33% for phagocytosis and 70% in the production of NO-). The rank correlation analysis revealed a significant, positive, and strong correlation (rho = 0.92) between NO- production in response to E. coli and S. aureus, and a positive but moderate correlation (rho = 0.58) between phagocytosis of E. coli and S. aureus. To gain further insight into this trait, another 58 cows were evaluated solely for NO- response against E. coli. The pedigree of the tested animals was added to the statistical model and the heritability was estimated to be 0.776. Overall, the finding of this study showed a strong effect of host genetics on the in vitro activities of MDM and the possibility of ranking Holstein cows based on the in vitro functional variation of MDM.

The effect of immunological status, in-vitro treatment and culture time on expression of eleven candidate reference genes in bovine blood mononuclear cells.

BACKGROUND: Technical feasibility of RNA quantification by real time RT-PCR has led to enormous utilization of this method. However, real time PCR results need to be normalized due to the high sensitivity of the method and also to eliminate technical variation. Normalization against a reference gene that is constitutively transcribed and has minimum variation among samples is the ideal method. Nevertheless, many studies have shown that there is no general reference gene(s) with ideal characteristics and candidate reference genes should be tested before being used as a "normalizer" in each study. METHODS: The current study investigated the effects of previous exposure of the host to experimental test antigens and culturing time on the expression of 11 candidate genes when blood mononuclear cells (BMCs) were cultured and treated in-vitro by hen egg white lysozyme, Candida albicans extract and a mitogen. Mononuclear cells were isolated and cultured from 12 bovine blood samples representing 3 different immunological statuses. The expression of candidate housekeeping genes were measured by real-time RT-PCR at 4 and 24 hours post culture. The expression of candidate genes were first compared between the two time points in untreated samples. Constitutively expressed genes were further tested in linear mixed effects models to examine the effect of previous host exposure and in-vitro treatments. RESULTS: Our findings showed that the expression of the most common reference genes, ß-actin, and Glyceraldehydes-3-phosphate dehydrogenase (GAPDH), are significantly decreased at 24 hours after culturing BMCs, even without any treatment. The effect of culturing time was also significantly influenced the expression of 18s ribosomal RNA, ß2-microglobulin, Tyrosine 3-monooxygenase/tryptophan 5-monoxygenase activation protein, zeta polypeptide (YWHAZ) in BMCs. Only the expression of C-terminal binding protein 1 (CTBP1) and RAD50 among all tested genes were consistent after treatment of cultured BMCs with C. albicans whole yeast extract and Hen Egg White Lysozyme (HEWL), respectively. In addition, expressions of CTBP1, and RAD50 were independent from previous exposure of the host to the antigen. CONCLUSIONS: The results of this study demonstrated inconsistent expression of commonly used reference genes in untreated cultured BMCs over time. As this condition applies to negative controls in real time RT-PCR study designs, normalization against these genes can largely deceive the outcome, especially in kinetic studies. Moreover, the potential effects of immunological memory on the expression of reference genes should be considered if BMCs are collected from different individuals under different environmental conditions and if these cells are treated in-vitro by an antigen.