Identifying Signaling Pathways and Biomarkers in Autoimmune Diseases

Laura Tabellini on June 24, 2019

As efforts in developing targeted therapies for autoimmune disease are rapidly advancing in basic research, scientists aim to validate immunology-relevant signaling pathways and uncover new intracellular molecules as potential drug targets.

The NanoString® nCounter® Autoimmune Profiling and Immunology panels are designed to rapidly analyze prominent aspects of autoimmune disease and aberrant immune responses. These panels and several Custom CodeSets have been featured in the literature to detect low expressing transcripts, regulatory pathway genes, interferon stimulated gene (ISGs) signatures, and novel regulatory mechanisms in various autoimmune diseases.

Interferonopathies and the Challenges of Low Expressing Markers

Type I interferon (IFN) and IFN-signaling are strongly implicated in the pathogenesis of numerous autoimmune inflammatory diseases and in interferonopathies. Upregulation of type I interferon triggers the expression of a broad signature of gene transcripts that reflect induction by IFNs. The detection of IFN remains challenging as it is usually present in low amounts in serum. Pescarmona and colleagues compared the NanoString nCounter platform’s multiplexing capabilities to traditional RT-qPCR in measuring IFN response in blood samples exhibiting type I interferonopathies.

The aims of this study were (1) to set up a test to measure the interferon response and (2) to compare the results obtained with two techniques: RT-qPCR and the NanoString nCounter platform. The group quantified the expression of six ISGs previously found to reflect the overall exposure to IFNs in Aicardi-Goutières syndrome, lupus and other interferonopathies. The group utilized a Custom CodeSet requiring only 200 ng of RNA. Compared to RT-qPCR, nCounter technology does not require reverse transcription or amplification of genetic material, hence eliminating the two main causes of bias with RT-qPCR. Furthermore, because NanoString assays can be used to multiplex different genes, other interesting target genes like IL-1β, IL-6 or TNF-α can be easily added to the assay with minimal effort. The correlation between these two methods was strong for each of the ISGs and thus for the IFN score. Moreover, ROC curves established with each technique in a pediatric cohort demonstrated high sensitivity and negative predictive value for the diagnosis of interferonopathies. In terms of analytical performance, while both methods achieved similar results, the NanoString nCounter platform was quicker, easier to multiplex, and nearly fully automated, making it an obvious and more reliable choice for future assay development.  

A Partner in Research

The molecular heterogeneity of autoimmune and inflammatory diseases remains a main obstacle to the development of safe and specific therapeutic options; thus, the need to find robust and actionable biomarkers has become paramount. Easy to customize, simple to use and to interpret, NanoString nCounter gene expression panels are pivotal in helping researchers decipher the heterogeneity behind complex diseases, allowing them to narrow in on potential biomarkers.

Sialic acid binding Ig like lectin 1 (SIGLEC-1) is a potential cell-type specific biomarker for identifying an overt type I IFN response. It mediates phagocytosis and endocytosis of pathogens, thereby promoting efficient immune responses to limit infection, making it an attractive marker for disease activity or drug response. It is expressed exclusively in cells of the myeloid lineage, namely tissue-resident macrophages and monocyte-derived dendritic cells. In blood, expression of surface SIGLEC-1 is restricted to CD14+ monocytes, therefore requiring intact cells and flow cytometry for its detection. A NanoString Custom CodeSet helped Oliveira and colleagues develop a novel immunoassay to detect circulating SIGLEC-1.

RNA from systemic lupus erythematosus (SLE) samples and age and sex-matched healthy donors was extracted from freshly isolated PBMCs. The authors hybridized 50 ng of total RNA with a Custom CodeSet containing probes for 56 interferon response genes previously identified as discriminative of the IFN signature. The authors compared SIGLEC-1 to other markers of disease activity, specifically the correlation between the SIGLEC-1 concentration and the canonical IFN transcriptional signature obtained through NanoString analysis. The group demonstrated that in combination with additional available IFN-regulated biomarkers, the SIGLEC-1 bioassay could improve the ability to dissect the molecular heterogeneity of the complex conditions associated with an overt IFN response and identify subsets of common and rare autoimmune and inflammatory diseases, collectively classified as interferonopathies.

In a paper published by Lundy and colleagues in 2018, the Human Immunology Panel was used to describe the dramatic shifts toward higher IFNγ production and cellular activation in the CD4 cell lineage of autoimmune retinopathy (AIR) samples. This disease causes rapidly progressive but treatable vision loss and is often confused with retinitis pigmentosa (RP) resulting in damaging delays in treatment. Currently, to determine if autoimmunity against retinal antigens is present, Western blot and histological assays have been developed as laboratory diagnostic tests to detect anti-retinal antibodies. However, it is critical to develop methods for early detection of AIR, and to have better defined options to preserve vision in these cases. To search for better actionable biomarkers, Lundy and colleagues designed a study to compare immune biomarkers and responses toward the retinal protein, recoverin, between a newly diagnosed AIR group, a slow progressing RP group and healthy controls.

RNA was extracted from whole blood collected in PAXgene tubes and the NanoString Human Immunology Panel was hybridized to the samples. Gene expression analysis revealed upregulation of mRNA for several genes involved in intracellular signaling and lymphocyte activation, particularly within CD4+ T helper cell lineage including: NFATc1, several STAT transcription factors associated with cytokine receptor signaling, integrin subunits involved in cell adhesion, and interferon response factors. These results show a Th1-mediated immune response as a common feature among those newly diagnosed with AIR.

A NanoString Custom CodeSet was used by Bauché and colleagues to demonstrate defects in mucosal immune tolerance leading to inflammatory bowel disease (IBD) such as Crohn’s disease and ulcerative colitis. Interleukin-22 (IL-22)-producing group 3 innate lymphoid cells (ILC3) maintain gut homeostasis but can also promote IBD. The authors sought to determine key mechanisms and regulation of ILC3-dependent colitis. They used a Custom CodeSet containing key autoimmune genes to profile direct lysate from FFPE tissue sections. The group demonstrated that LAG-3+ Treg cells suppress the production of proinflammatory cytokines by CX3CR1+ macrophages and thereby inhibit ILC3-driven colitis.

From Mouse Models to Humans

Animal models are pivotal to understanding autoimmune diseases, as demonstrated by a large body of peer reviewed papers reflecting the advances brought by these models in terms of deciphering disease mechanisms. Plus, the relative lack of progress in human autoimmune diseases for which there is no animal model further corroborates the important role of animal models.

For this purpose, Mande and colleagues developed a mouse model of systemic autoimmunity and used the NanoString PanCancer Immune Profiling panel as a discovery tool to demonstrate  the relevance of their mouse model in studying human lupus-like symptoms. The group aimed to investigate the negative regulatory role of TLR9 in cutaneous lupus erythematosus (CLE), a dermatologic feature of SLE.

The authors developed an inducible rapid-onset murine model of systemic autoimmunity that depends on T cell detection of a membrane-bound OVA fusion protein expressed by MHC class II cells, expression of TLR7, expression of the type I IFN receptor, and loss of expression of TLR9. The group profiled 100 ng of RNA from mouse skin biopsies using the mouse PanCancer Immune Profiling panel. They followed up with validation work in human samples of lesioned skin from subjects with lupus or psoriasis: 100 ng of RNA was profiled using the human PanCancer Immune Profiling panel; gene expression profiles were compared to those of healthy controls.

Results demonstrated overlapping genes deregulated in both the human and mouse model highlighting the strength of NanoString’s nCounter platform as a discovery tool: many of the ISGs and chemokines highly upregulated in both the mouse model and in human lupus lesions and, to a lesser extent, in psoriasis lesions, further support the relevance of the mouse model to human lupus-like symptoms. Importantly, of all the genes investigated, FasL was found to be the key effector mechanism in the skin in mouse models, as the transfer of FasL-deficient DO11gld T cells completely failed to elicit overt skin lesions. FasL was also upregulated in human CLE biopsies. Overall, the authors’ model provides a relevant system for exploring the pathophysiology of lupus as well as the negative regulatory role of TLR9.

FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.


Pescarmona et al., Cytokine. 2019 Jan;113:446-452

Oliviera et al., Arthritis Res Ther. 2018 Jul 27;20(1):152

Lundy et al., Front Med (Lausanne). 2018 Sep 13;5:249

Bauché D. et al., Immunity. 2018 Aug 21;49(2):342-352.e5

Mande et al., J Clin Invest. 2018 Jul 2;128(7):2966-2978

Post by Laura Tabellini