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Significance Inflammation has largely been studied in the context of protein-coding genes. Recent studies have uncovered lncRNAs as important regulators of immunity. The functional characterization of these genes remains an active area of research. In this study, we identify GAPLINC as a functionally conserved lncRNA between human and mouse. GAPLINC depletion results in enhanced expression of immune response genes that are direct NF-κB targets. Astoundingly, we observe that Gaplinc knockout mice show resistance to LPS-induced endotoxic shock and find that basal expression of inflammatory genes prevents clot formation to protect against multiorgan failure and death. These findings have implications in the treatment of sepsis, in which new therapies targeting lncRNAs can contribute valuable information in understanding inflammation and improving patient outcome., Recent studies have identified thousands of long noncoding RNAs (lncRNAs) in mammalian genomes that regulate gene expression in different biological processes. Although lncRNAs have been identified in a variety of immune cells and implicated in immune response, the biological function and mechanism of the majority remain unexplored, especially in sepsis. Here, we identify a role for a lncRNA—gastric adenocarcinoma predictive long intergenic noncoding RNA (GAPLINC)—previously characterized for its role in cancer, now in the context of innate immunity, macrophages, and LPS-induced endotoxic shock. Transcriptome analysis of macrophages from humans and mice reveals that GAPLINC is a conserved lncRNA that is highly expressed following macrophage differentiation. Upon inflammatory activation, GAPLINC is rapidly down-regulated. Macrophages depleted of GAPLINC display enhanced expression of inflammatory genes at baseline, while overexpression of GAPLINC suppresses this response. Consistent with GAPLINC-depleted cells, Gaplinc knockout mice display enhanced basal levels of inflammatory genes and show resistance to LPS-induced endotoxic shock. Mechanistically, survival is linked to increased levels of nuclear NF-κB in Gaplinc knockout mice that drives basal expression of target genes typically only activated following inflammatory stimulation. We show that this activation of immune response genes prior to LPS challenge leads to decreased blood clot formation, which protects Gaplinc knockout mice from multiorgan failure and death. Together, our results identify a previously unknown function for GAPLINC as a negative regulator of inflammation and uncover a key role for this lncRNA in modulating endotoxic shock.

Determining the layers of gene regulation within the innate immune response is critical to our understanding of the cellular responses to infection and dysregulation in disease. We identified a conserved mechanism of gene regulation in human and mouse via changes in alternative first exon (AFE) usage following inflammation, resulting in changes to isoform usage. Of these AFE events, we identified 50 unannotated transcription start sites (TSS) in mice using Oxford Nanopore native RNA sequencing, one of which is the cytosolic receptor for dsDNA and known inflammatory inducible gene, Aim2. We show that this unannotated AFE isoform of Aim2 is the predominant isoform transcribed during inflammation and contains an iron-responsive element in its 5′UTR enabling mRNA translation to be regulated by iron levels. This work highlights the importance of examining alternative isoform changes and translational regulation in the innate immune response and uncovers novel regulatory mechanisms of Aim2.Summary SentenceAlternative first exon usage was the major splicing event observed in macrophages during inflammation, which resulted in the elucidation of a novel isoform and iron mediated regulatory mechanism of the protein coding gene, Aim2.

Background The presence of antibodies against cyclic citrullinated proteins (anti-CCP) precede the development of rheumatoid arthritis (RA) and both anti-CCP antibody and RF positivity indicate more severe disease. This characteristic makes Anti-CCP best biomarker candidate for the prevention of debilitating joint disease. Almost 70% of RA patients are positive for anti-CCP IgG, while only 2% of control subjects are positive for anti-CCP. Understanding the origins of anti-CCP is critical in unraveling the etiology of RA, so we further evaluated the association of the other autoimmune tests/biomarkers with Anti-CCP to describe other comorbid autoimmune diseases/syndrome. Methods We conducted a retrospective analysis of patient data from April 18, 2016, to May 29, 2019, in Texas. The study protocol was approved by the ethics review committee and patients consented for sharing their demographics, clinical information and lab values for diagnostic and research purposes. The study population was described by descriptive statistics. Demographic characteristics and laboratory parameters were compared using chi-square tests. We analyzed blood tests/parameters conducted at the clinics and explored their association with the presence of ACCP. logistic regression models were utilized to calculate the odds ratio to delineate the strength of association of different blood parameters with ACCP. IgG anti-CCP was determined according to the manufacturer’s instruction. Results Eleven thousand, four hundred fifty-nine tests were performed on 7,200 patients with a mean age of 52 years (range 0-98). Almost half (54%) of study individuals were females, two-third (63%) were White, (29%) Hispanic and (7%) African American. Four hundred and six patients (3.5%) were positive for AntiCCP3.1. Among those who were positive for AntiCCP3.1, (22%) were Rh factor IgA (RhIgA) positive, (11%) RhIgG positive, and (9%) RhIgM. Univariate logistic regression showed that the female gender was positively associated (OR = 3.19) with ACCP presence while patients older than 50 had a positive gradient of ACCP positivity with each age category. Compared to White, African Americans were more likely (OR = 3.19) and Hispanics were less likely (OR = 0.44) to be associated with AntiCCP positivity. After controlling for age, gender and ethnicity the presence of RhIgM and RhIgA antibodies were positively associated with positive ACCP. (OR = 13.3, OR = 5.2). antiphosphatidylserine IgM and anticardiolipin IgM antibodies were positively associated with ACCP (OR = 2.1, OR = 3.4). Similarly, the presence of antimitochondrial M2 antibodies and anti-topoisomerase antibodies (SCL-70) were very significantly associated with ACCP (OR = 16.1, OR = 37.8). Conclusion The Anti-CCP antibodies are highly specific for the early diagnosis of RA and with early initiation of treatment, progression to irreversible and destructive diseases can be prevented. Association of Anti-CCP antibodies with other autoimmune antibodies highlights the importance of screening RA patients for primary biliary sclerosis and scleroderma. Similarly, a biomarker's strength of interaction with another biomarker helps in the prognostic utility of these test for preventing co-existing diseases. Disclosures M. Ayass: Other; Physician at Ayass Bioscience. K. Zhu: Other; Employee at Ayass Bioscience. S. Naveed: None. L. Mosleh: Other; Employee at Ayass Bioscience. K. Viswanathan: Other; Employee at Ayass Bioscience. G. Nowshad: Other; Employee at Ayass Bioscience.

Background Systemic lupus erythematosus (SLE) is characterized by the presence of multiple autoantibodies as well as clinical evidence of multi-system immune-mediated pathology. Individuals who develop SLE typically demonstrate serological autoimmunity for several years prior to the onset of clinical disease. Measuring anti-nuclear antibodies (ANA) is a common test for humoral autoimmunity. It is sensitive (95+%) for SLE, but not specific as up to 25% of healthy controls (HC) will have a measurable ANA. While very few of the ANA +HC will develop SLE, they represent a group at higher risk of the disease. Understanding the risks for ANA positivity provides essential knowledge about the development of SLE. Methods Serum and DNA were collected from 2903 healthy individuals with no personal history of autoimmunity. Antinuclear antibodies were detected using Inova QuantaLite ELISA. Sera from subset (n=724) individuals (ANA− HC, ANA+ HC, and SLE) were assayed by protein microarray quantifying IgM and IgG responses to 89 previously known human autoantigens. A nested cohort consisting of all the ANA +Caucasian individuals and age/gender matched ANA− controls were genotyped using the ImmunoChipv.1 SNP array. Results In HC, 16% had moderate and 10% had high levels of IgG ANA. Autoantigen microarray data showed that ANA+ HC had a high prevalence of antibodies to non-nuclear and cytoplasmic antigens while subjects with SLE predictably produced antibodies to a variety of nuclear antigens as well. A quantitative genetic association test with ANA identified the locus c11orf30 or EMSY, associated with high ANA phenotype in the healthy population (see figure 1). This locus codes for a negative transcriptional regulator. A haplotype comprised of many potentially regulatory polymorphisms at EMSY contributed to strong risk for ANA in healthy individuals [(p=3.83E-04, OR=2.6)]. eQTL data suggests that the ANA-associated EMSY haplotype leads to reduced expression of EMSY protein in human macrophages and EBV cell lines. Autoantibody profiles of serum samples from healthy individuals with the EMSY risk genotype exhibited high titers of anti-IgG and IgA antibodies targeted to multiple autoantigens as well as food and environmental allergens. Conclusions EMSY, a locus previously linked with atopy, psoriasis and inflammatory bowel disease was associated with ANA in healthy individuals. The EMSY protein is a BRCA2-associated transcriptional repressor. Individuals with risk haplotypes in EMSY make a wide variety of disease-associated antibodies, suggesting an early common pathway for autoimmune and allergic conditions.

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disease with extensive clinical heterogeneity, affecting mainly females. It is initiated with breach in immune tolerance and as a result one of the main characteristics of SLE is the production of autoantibodies to a wide spectrum of self-antigens. Sle1 is a potent susceptibility locus for spontaneous systemic autoimmunity derived from the NZM2410 mouse strain. The NZW-derived suppressive modifier locus, Sles1, specifically prevents the spontaneous loss in tolerance mediated by the B6.Sle1 congenic. Fine mapping combined with phenotypic characterization of series of recombinant mouse strains have mapped Sles1 to an approximately 638 Kb segment on murine chromosome 17. This revealed extensive epistatic gene interactions within the Sles1 region and suggested Btnl2 and H2 genes as the strongest candidates. We generated BAC transgenic mice by introducing BAC harboring B6 allele of Btnl2 alone or Btnl2 and H2 combined allelic region on to Sle1.Sles1 mice to identify the gene driving the suppressive phenotype. Phenotypic characterization of aging BAC transgenic mice is completed and results strongly indicate Btnl2 as the key gene within the 638 Kb region driving the suppression. Additionally we have performed targeted resequencing on B6 and B6.Sles1 mice and have identified functional polymorphisms within the Btnl2 gene between the two mice strains. We are currently generating and analyzing RNA-seq data on our BAC transgenic mice to gain insight in to the mechanisms on how Btnl2 is suppressing autoimmunity. This study confirms Btnl2 as the key driver gene in suppressing loss of tolerance in SLE pathogenesis.

The human immune system defends the body against microbes and other threats. However, if this process goes wrong the immune system can attack the body’s own healthy cells, which can lead to serious autoimmune diseases. Systemic lupus erythematosus (SLE) is an autoimmune disease in which immune cells often attack internal organs – including the kidneys, nervous system and heart. Over the past decade, multiple genes have been linked with an increased risk of SLE. However, it is largely unknown how the sequences of these genes differ between individuals with SLE and healthy individuals, and the precise changes that lead to an increased risk of SLE are also not clear. Now, Raj, Rai et al. have determined the genetic sequences of over 700 people with SLE and over 500 healthy individuals and looked for differences that influence susceptibility to the disease. The vast majority of differences were discovered in stretches of DNA that regulate the expression of nearby genes, rather than in DNA that encodes the structures of proteins. Notably, extensive differences were found in a region of the human genome that regulates the production of proteins called Human Leukocyte Antigen class II molecules; which are known to play a critical role in activating the immune system. Raj, Rai et al. found that slight changes to the regulatory DNA sequences resulted in an overabundance of these proteins, which led to a hyperactive immune system that is strongly associated with SLE. Future studies could now ask if the changes to the regulatory DNA sequences highlighted by Raj, Rai et al. increase susceptibility to other autoimmune disorders as well. It may also be possible to use the increased understanding of how the immune system is regulated to develop new ways to minimize the rejection of organ transplants.

The Toll-like receptors (TLR) of the innate immune system play a key role in the recognition of pathogens and the initiation of a robust innate immune response. Although an innate immune response is essential for resistance to pathogen infection, the magnitude and qualities of innate immune responses are quite variable in the human population. Here we performed transcriptome sequencing of monocyte-derived macrophages generated from 78 healthy individuals, including 10 replicates from repeat donors, to profile gene expression patterns in responses to TLR7/8 signaling. Extensive qualitative and quantitative diversity was apparent in the response of individuals in this panel. Cluster analysis discriminated nine distinct clusters of genes expressed in response to the TLR7/8 agonist (R848) with correlated expression variations within this panel. Interestingly, the induction of cytokine and chemokine transcription varied among donors and majority of these genes have very reproducible variability of their expression in 10 replicates. In addition, it was shown that donors with high expression of cytokine and chemokine genes after R848 stimulation were also high responder to Respiratory syncytial virus infection. Thus, there are the variations in gene expression among individuals with TLR7/8 stimulation, indicating that genetic polymorphisms in “master” regulators may correlate expression patterns of multiple genes during innate responses.

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease in infants. Little is known about the cell-intrinsic innate immune responses of RSV infected epithelial cells or responding immune cells. We studied two clinical-isolates from the RSV-B subgroup: NH1125 that induces high cytokine responses (measured by CCL5 and IL-6); and NH1067, which induces low cytokine responses. We used RNA sequencing technologies to profile the transcriptome of non-immune and immune-lineage cells following infection with these two strains. The innate immune response of A549, a human epithelial cell line, was distinct from that of monocyte-derived macrophages and dendritic cells at 2, 4, 8, and 24 hours following infection. These responses were compared at a high multiplicity of infection (MOI=5) for a homogeneous response and low MOI (MOI=0.3), which more closely resembles in vivo conditions, to determine the extent of communication between infected and un-infected cells. Our analyses revealed 6 classes of genes marking the early- and late-stage cellular responses. Pathway analyses identified miRNA let-7f, EIF2 signaling and mitochondrial dysfunction signaling as early activated pathways and pattern-recognition receptor and Interferon pathway at later time points. A cross-comparison also reveals that RSV gene expression and viral replication is greater in A549 cells than macrophages, consistent with a more potent cell-intrinsic innate defense in myeloid lineage cells.