Longitudinal saliva omics responses to immune perturbation: a case study.

Saliva omics has immense potential for non-invasive diagnostics, including monitoring very young or elderly populations, or individuals in remote locations. In this study, multiple saliva omics from an individual were monitored over three periods (100 timepoints) involving: (1) hourly sampling over 24 h without intervention, (2) hourly sampling over 24 h including immune system activation using the standard 23-valent pneumococcal polysaccharide vaccine, (3) daily sampling for 33 days profiling the post-vaccination response. At each timepoint total saliva transcriptome and proteome, and small RNA from salivary extracellular vesicles were profiled, including mRNA, miRNA, piRNA and bacterial RNA. The two 24-h periods were used in a paired analysis to remove daily variation and reveal vaccination responses. Over 18,000 omics longitudinal series had statistically significant temporal trends compared to a healthy baseline. Various immune response and regulation pathways were activated following vaccination, including interferon and cytokine signaling, and MHC antigen presentation. Immune response timeframes were concordant with innate and adaptive immunity development, and coincided with vaccination and reported fever. Overall, mRNA results appeared more specific and sensitive (timewise) to vaccination compared to other omics. The results suggest saliva omics can be consistently assessed for non-invasive personalized monitoring and immune response diagnostics.

Hypoxia-Inducible Factor (HIF)-1α Promotes Inflammation and Injury Following Aspiration-Induced Lung Injury in Mice.

Acid aspiration-induced lung injury is a common disease in the intensive care unit (ICU) and acute respiratory distress syndrome (ARDS). Hypoxia-inducible factor (HIF)-1α is a major transcription factor responsible for regulating the cellular response to changes in oxygen tension. A clear understanding of the function of HIF-1α in lung inflammatory response is currently lacking. Here, we sought to determine the role of HIF-1α in type 2 alveolar epithelial cells (AEC) in the generation of the acute inflammatory response following gastric aspiration (GA). GA led to profound hypoxia at very early time points following GA. This correlated to a robust increase in HIF-1α, tissue albumin and pro-inflammatory mediators following GA in AECs. The extent of lung injury and the release of pro/anti-inflammatory cytokines were significantly reduced in HIF-1α (-/-) mice. Finally, we report that HIF-1α upregulation of the acute inflammatory response is dependent on NF-κB following GA.

RNA-seq Analysis Reveals Unique Transcriptome Signatures in Systemic Lupus Erythematosus Patients with Distinct Autoantibody Specificities.

Systemic lupus erythematosus (SLE) patients exhibit immense heterogeneity which is challenging from the diagnostic perspective. Emerging high throughput sequencing technologies have been proved to be a useful platform to understand the complex and dynamic disease processes. SLE patients categorised based on autoantibody specificities are reported to have differential immuno-regulatory mechanisms. Therefore, we performed RNA-seq analysis to identify transcriptomics of SLE patients with distinguished autoantibody specificities. The SLE patients were segregated into three subsets based on the type of autoantibodies present in their sera (anti-dsDNA+ group with anti-dsDNA autoantibody alone; anti-ENA+ group having autoantibodies against extractable nuclear antigens (ENA) only, and anti-dsDNA+ENA+ group having autoantibodies to both dsDNA and ENA). Global transcriptome profiling for each SLE patients subsets was performed using Illumina® Hiseq-2000 platform. The biological relevance of dysregulated transcripts in each SLE subsets was assessed by ingenuity pathway analysis (IPA) software. We observed that dysregulation in the transcriptome expression pattern was clearly distinct in each SLE patients subsets. IPA analysis of transcripts uniquely expressed in different SLE groups revealed specific biological pathways to be affected in each SLE subsets. Multiple cytokine signaling pathways were specifically dysregulated in anti-dsDNA+ patients whereas Interferon signaling was predominantly dysregulated in anti-ENA+ patients. In anti-dsDNA+ENA+ patients regulation of actin based motility by Rho pathway was significantly affected. The granulocyte gene signature was a common feature to all SLE subsets; however, anti-dsDNA+ group showed relatively predominant expression of these genes. Dysregulation of Plasma cell related transcripts were higher in anti-dsDNA+ and anti-ENA+ patients as compared to anti-dsDNA+ ENA+. Association of specific canonical pathways with the uniquely expressed transcripts in each SLE subgroup indicates that specific immunological disease mechanisms are operative in distinct SLE patients' subsets. This 'sub-grouping' approach could further be useful for clinical evaluation of SLE patients and devising targeted therapeutics.

Differential clearance mechanisms, neutrophil extracellular trap degradation and phagocytosis, are operative in systemic lupus erythematosus patients with distinct autoantibody specificities.

Systemic lupus erythematosus (SLE) patients are generally presented with autoantibodies against either dsDNA or RNA-associated antigens (also known as extractable nuclear antigens, ENA) or both. However, the mechanisms and processes that lead to this distinctive autoantibody profile are not well understood. Defects in clearance mechanism i.e. phagocytosis may lead to enhanced microbial and cellular debris of immunogenic potential. In addition to defective phagocytosis, impaired neutrophil extracellular trap (NET) degradation has been recently reported in SLE patients. However, the extent to which both these clearance processes (NET-degradation and phagocytosis) are operative in serologically distinguished subsets of SLE patients is not established. Therefore, in this report, we evaluated NET-degradation and phagocytosis efficiency among SLE patients with different autoantibody specificities. SLE patients were classified into three subsets based on their autoantibody profile (anti-dsDNA, anti-ENA or both) as determined by ELISA. NET-degradation by SLE and control sera was assessed by sytox orange-based fluorescence assay. Neutrophil-mediated phagocytosis in the presence of SLE and control sera was determined by flowcytometry. The segregation of SLE patients revealed significant differences in NET-degradation and phagocytosis in SLE patients with autoantibodies against dsDNA and ENA. We report that NET-degradation efficiency was significantly impaired in SLE patients with anti-dsDNA autoantibodies and not in those with anti-ENA autoantibodies. In contrast to NET-degradation, neutrophil-mediated phagocytosis was impaired in all three subsets independent of autoantibody specificity. These observations suggest that varying clearance mechanisms are operative in SLE subsets with anti-dsDNA or anti-ENA autoantibodies. The results outlined in this manuscript also suggest that sub-grouping of SLE patients could be useful in delineating the molecular and pathological processes that are often missed when SLE patients are studied as a single group. Further, it will be imperative to propose that therapies targeted at improving NET clearance can be effective in anti-dsDNA(+) SLE patients.

Heat shock protein 27 and its regulatory molecules express differentially in SLE patients with distinct autoantibody profiles.

Generation of autoantigens of nuclear origin, like dsDNA and extractable nuclear antigens (ENA) have largely been associated with dysregulated apoptosis and defective clearance of apoptotic debris in SLE. Heat shock protein (HSP) 27 has been reported to have anti-apoptotic properties hence it was of interest to study the expression of HSP27 and its regulatory molecule Brn3a and hsa-miR-939 in SLE patients with distinct autoantibodies specificities. SLE patients were categorized into three subsets based on their distinct sero-positivity for either anti-dsDNA antibody alone (anti-dsDNA(+) group) or anti-ENA antibody alone (anti-ENA(+) group) or both (anti-dsDNA(+) ENA(+) group). We investigated the mRNA and protein expression of HSP27 and Brn3a in peripheral blood leukocytes (PBLs) by real-time reverse transcriptase PCR and Western blotting. Expression of apoptosis markers caspase 3 and poly (ADP-ribose) polymerase (PARP) was determined by Western blotting. Hsa-miR-939 expression was determined using TaqMan(®) miRNA assay. In this study, we report significant downregulation of HSP27 in anti-ENA(+) patients and increased expression of caspase 3 and PARP in both anti-ENA(+) and anti-dsDNA(+) SLE subsets. A negative correlation was observed between the expression of HSP27 and apoptosis markers caspase 3 and PARP. Decreased Brn3a expression was observed in anti-ENA(+) SLE patients, which correlated positively with HSP27 expression. Expression of hsa-miR-939, which has a potential target site for Brn3a 3' UTR, was also elevated specifically in anti-ENA(+) patients. The decreased expressions of HSP27, Brn3a along with elevated levels of hsa-miR-939 are selectively associated with anti-ENA(+) patients and HSP27 was observed to be inversely associated with apoptosis. These findings are suggestive of distinct regulatory processes operative in SLE patient subsets with different autoantibody specificities.

Decreased toll-like receptor-4/myeloid differentiation factor 88 response leads to defective interleukin-1ß production in term low birth weight newborns.

BACKGROUND: Morbidity and mortality rates are very high in low birth weight (LBW) newborns because of their increased susceptibility to infections compared with normal birth weight (NBW) newborns. A case and control study was designed to identify the status of toll-like receptor-4 (TLR-4) signaling and maternally derived immunoglobulin-G (IgG) subclasses in term LBW newborns compared with NBW newborns. METHODS: To understand the basis of increased susceptibility to infections in LBW newborns, the levels of pro- and antiinflammatory cytokines interleukin-1ß (IL-1ß) and interleukin-10 (IL-10), respectively, released in response to lipopolysaccharide (LPS) stimulation of cord blood cells of LBW (n = 20) and NBW (n = 18) newborns, were quantified by enzyme-linked immunosorbent assay. Further, LPS-induced expression of TLR-4 and basal and LPS-induced expression of myeloid differentiation factor 88 (MyD88) were examined at mRNA levels in both groups. The levels of IgG subclasses in LBW (n = 20) and NBW (n = 18) newborns were quantified by enzyme-linked immunosorbent assay to explore the role of maternally derived immunity in LBW newborns. RESULTS: LPS-mediated release of IL-1ß was significantly diminished in LBW newborns when compared with NBW newborns, whereas there was no significant difference in IL-10. Decreased production of IL-1ß in LBW newborns was correlated with reduced expression of TLR-4 and MyD88 mRNA. No significant differences were observed in the levels of all 4 IgG subclasses between LBW and NBW newborns. CONCLUSIONS: Decreased production of IL-1ß in LBW newborns was correlated with reduced expression of TLR-4 and MyD88 mRNA. This raises the possibility of increased susceptibility to infections in LBW when compared with the NBW newborns at term. Comparable levels of IgG subclasses in the 2 groups of newborns indicate that IgG is not a limiting factor in defense against infection in LBW newborns.

Differential microRNA profile and post-transcriptional regulation exist in systemic lupus erythematosus patients with distinct autoantibody specificities.

PURPOSE: Systemic lupus erythematosus (SLE) patients have anti-nuclear autoantibodies directed against dsDNA and RNA-associated antigens (extractable nuclear antigens; ENA). In this study, we investigated the differences in microRNA (miRNA) expression and its biological implications in SLE patients with distinct autoantibody specificities. METHODS: The SLE patients were grouped into three subsets based on the type of autoantibodies present in their sera (anti- ENA+ group with autoantibodies against ENA alone; antidsDNA+ group having autoantibodies against dsDNA only, and anti-ENA+dsDNA+ group having autoantibodies to both dsDNA and ENA). Global miRNA expression profiling was done for each of these three groups using TaqMan® low density miRNA arrays. RESULTS: We report that different sets of miRNAs are dysregulated in SLE patients with different autoantibody specificities. Further, Ingenuity pathway analysis (IPA) software revealed specific biological pathways that were targeted by miRNAs dysregulated in different SLE subsets. Molecules involved in cell cycle and cytoskeleton remodeling were the prime targets of miRNAs dysregulated in anti-ENA+ patients whereas miRNAs dysregulated in anti-dsDNA+ patients were found to be implicated in multiple cytokine signaling pathways. IPA analysis of gene targets of miRNAs commonly dysregulated in all three SLE subsets identified several metabolic-, hormone-, and interferon-related pathways to be affected. CONCLUSION: The differential miRNA expression in patients with distinct autoantibodies is suggestive of different regulatory mechanisms operating among them. Based on these observations, we are hopeful that this 'sub-grouping' approach could be used to identify other defective processes associated with varying disease manifestations in SLE and may be considered when designing therapeutic interventions.

Distinct autoantibody profiles in systemic lupus erythematosus patients are selectively associated with TLR7 and TLR9 upregulation.

PURPOSE: Systemic lupus erythematosus (SLE) patients have a wide array of autoantibodies against nuclear antigens. The two predominant classes of these autoantibodies are directed either against dsDNA or RNA-associated antigens (extractable nuclear antigens; ENA). Nucleic-acid sensing Toll-like receptors (TLRs) that recognize dsDNA and RNA, have been well implicated in some murine models of SLE. We took up this study to identify if unique TLR expression patterns are associated with distinct autoantibody profiles in SLE. METHODS: We segregated the patients into three subsets distinguished on the basis of autoantibody response either against dsDNA or ENA or both. We determined the mRNA expression of TLR3, 7, 8, and 9 by real-time reverse-transcription PCR in peripheral blood leucocytes (PBLs) of the SLE patients of all three subsets. TLR7 and 9 protein expression was determined by western blotting in PBLs and by flow cytometry on B-cells and monocytes. The serum interferon-alpha (IFN-α) and anti-dsDNA/-ENA autoantibodies were detected using enzyme-linked immunosorbant assay. RESULTS: We report differential and unique TLR expression patterns associated with different autoantibody profiles. The presence of anti-ENA and anti-dsDNA autoantibodies in SLE patients was associated with elevated levels of TLR7 and TLR9 respectively. The TLR9 mRNA expression was further augmented in SLE patients with Glomerulonephritis. Interestingly, anti-dsDNA(+) ENA(+) patients displayed higher serum IFN-α and interferon regulatory factor 7 mRNA expression than patients with either anti-dsDNA or anti-ENA autoantibodies alone. CONCLUSION: Characteristic TLRs expression profile associated with distinct autoantibody repertoire is suggestive of differential immuno-regulatory pathways operative in different subsets of SLE patients.

Decreased pattern recognition receptor signaling, interferon-signature, and bactericidal/permeability-increasing protein gene expression in cord blood of term low birth weight human newborns.

BACKGROUND: Morbidity and mortality rates of low birth weight (LBW) newborns at term are higher than rates in normal birth weight (NBW) newborns. LBW newborns are at greater risk to acquire recurrent bacterial and viral infections during their first few weeks of life possibly as an outcome of compromised innate immune functions. As adaptive immunity is in a naive state, increased risk of infection of LBW as compared to NBW newborns may reflect impairments in innate immunity. METHODOLOGY: To characterize the increased susceptibility to infections in LBW newborns we used microarray technology to identify differences in gene expression in LBW newborns (n = 8) compared to NBW newborns (n = 4) using cord blood. The results obtained from the microarray study were validated on a larger number of samples using real time RT-PCR (LBW = 22, NBW = 18) and western blotting (LBW = 12, NBW = 12). The Interferome database was used to identify interferon (IFN) signature genes and ingenuity pathway analysis identified canonical pathways and biological functions associated with the differentially expressed genes in LBW newborns. ELISAs for IFNs and bactericidal/permeability-increasing protein were performed in both LBW and NBW newborns and in adults (LBW = 18, NBW = 18, Adults= 8). PRINCIPAL FINDINGS: Upon microarray analysis, we identified 1,391 differentially expressed genes, of which, 1,065 genes were down-regulated and 326 genes were up-regulated in the LBW compared to NBW newborns. Of note, 70 IFN-signature genes were found to be significantly down-regulated in LBW compared to NBW newborns. Ingenuity pathway analysis revealed pattern recognition receptors signaling including Toll-Like Receptors (TLRs) -1, -5, and -8 genes and IFN signaling as the most significantly impacted pathways. Respiratory infectious diseases were the most significantly affected bio-functions in LBW newborns. CONCLUSION AND SIGNIFICANCE: Diminished PRRs, IFN-signature, and BPI gene expression raises the possibility that impairments in these pathways contribute to the susceptibility of LBW term infants to infection.