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Small RNAs and immunological reactions

Patients with common variable immunodeficiency (CVID) lack antibodies that would protect them against infections. Only a handful of patients develop the disease because of a single defect in any of the genes involved in CVID pathogenesis. A group of researchers led by Dr. Ulrich Salzer at the Centre for Chronic Immunodeficiency (CCI) at the University of Freiburg Medical Centre investigates how small RNA molecules control B-lymphocyte development as well as potential defects. It is becoming increasingly clear that the immune system cells make complex finely tuned decisions rather than black and white ones.

The schematic shows the number of different cell types in healthy blood (top) and CVID patients (bottom). CVID patients have a much lower number of antibody-producing B cells (blue circle). © Dr. med. Ulrich Salzer
In contrast to many congenital immune system disorders, common variable immunodeficiency disease (CVID) usually occurs in adults rather than young children. Patients with CVID lack antibodies that would normally protect them against bacterial or viral intruders. The disorder is characterised by the lack of B lymphocytes (B cells) that are capable of producing antibodies, amongst other things. Patients with CVID frequently suffer from recurrent respiratory infections and pneumonia; autoimmunological phenomena and tumours have also been found to be associated with CVID. “CVID presents with a heterogeneous disease phenotype,” said Dr. Ulrich Salzer, head of a group of researchers at the Centre for Chronic Immunodeficiency (CCI) at the Freiburg University Medical Centre, which is funded by the German Ministry of Education and Research (BMBF). “As patients with CVID can develop a broad range of different clinical symptoms, the disease is often difficult to diagnose and treat.”

Control switches in the cell

In addition to being difficult to diagnose, little is yet known about the genetics and inheritance of CVID. Some gene defects and mutations have been identified and associated with CVID pathogenesis, including the TACI gene, which encodes a receptor protein on the surface of B cells and was identified by Ulrich Salzer during his postdoctoral period. However, the TACI gene and potentially also other genes are usually only seen as risk factors that increase susceptibility to CVID. Only a handful of CVID patients develop the disease due to a single defect in any of the hereditary factors involved in CVID pathogenesis.

In addition, there is increasing evidence that only a small proportion of CVID diseases have a monogenic cause. In order to shed light on CVID pathogenesis, Ulrich Salzer started concentrating on small RNA molecules, which are also found to play important regulatory roles in biological processes in other medical areas (e.g. oncology).

These small RNA molecules only consist of a small number of nucleotides and are referred to as microRNAs. Their sequences are complementary to certain sequences within mRNAs (messenger RNAs) that are translated into proteins. The binding of a microRNA molecule to such a sequence might disturb the translation of mRNA into the amino acid polymer, with the result that less protein is produced. “By binding to sequences within mRNAs, microRNAs thus indirectly regulate the transcription rate of genes. They can be seen as switches that control many cellular processes,” said Salzer.

From cancer research it has been known for some years that defects in these regulation processes may have an effect on tumour development. However, this possibility had not previously been taken into account for CVID. Research into the role of microRNAs in CVID development was triggered by research findings that suggested that microRNAs regulate gene expression and play a role in B-lymphocyte development.

Since 2009, the investigations of Salzer and his team have been carried out under the aegis of SFB 620 (collaborative research centre funded by the German Research Foundation, DFG) in a subproject looking into whether the B lymphocytes of CVID patients express microRNAs that are different from those expressed in the B lymphocytes of healthy individuals. Salzer’s investigations are based on the idea that defects in B-cell development might be due to the presence or absence of certain microRNAs. Salzer’s team carried out numerous screens and came up with around two dozen promising candidates, including a cluster of several microRNAs that until then had not been associated with B-cell development. Salzer and his team are currently investigating the potential function of this cluster in B cells.

Not a black and white process

How a reporter gene works: the cells shown in the picture are so-called Hela cells that are easier to look at under the microscope than B cells. The reporter construct consists of a GFP (green fluorescent protein) gene fused to a DNA sequence of the target mRNA with microRNA binding sites. Cells light up green when the construct is translated into a protein. Cells appear red and blue when the translation of the mRNA is inhibited by a microRNA. The red dye (WGA) stains the cell membrane, the blue (DAPI) dye cell nuclei. Using such a reporter gene system, researchers can find out whether a microRNA blocks a mRNA and hence its translation into protein when cells lose their green colour. The red/blue cells shown in the picture are evidence of the unsuccessful incorporation of the reporter gene. © Dr. med. Ulrich Salzer

The investigation of the potential function of this cluster will not be easy as the microRNA target structures (i.e. messenger RNAs) usually have numerous microRNA binding sites. It can be safely assumed that the regulatory process is rather complex and that it will most likely be impossible to relate a single microRNA with an up- or downregulated gene. “We assume that it is a combination of different erroneously expressed microRNAs and their target genes that leads to the development of one or other clinical CVID signs,” said Salzer. “We assume that microRNAs do not regulate cellular development in a black and white process, but instead they refine the tuning of processes at different sites in the cell.” Although they are working on a challenging issue, the Freiburg researchers have already identified several transcription factors that are controlled by microRNAs and that are in some way or other associated with the development of B cells.

The researchers will also use bioinformatic analysis methods in order to find all the players involved in the processes under investigation. Once these molecules have been identified, the researchers will study the role of the individual molecules that are specifically involved in B-cell development, for example by using knock-down experiments using inhibitors or by overexpressing the respective genes in healthy cells using reporter genes that enable the determination of microRNA-mediated regulation using fluorescent proteins such as GFP (green fluorescent protein) or luciferase (see figure on the right-hand side). 

How do the cells react in the absence of certain microRNAs or in the presence of higher than normal microRNA concentrations? Do they still produce antibodies? Salzer and his team might eventually be able to improve the diagnosis of certain patients and patient groups. Knowledge about the concrete causes of immunological disorders will enable predictions to be made about the progression of such diseases and their different symptoms. Such knowledge might also be of great importance to basic researchers as the finding that cellular processes are governed at levels other than the genetic level is relatively new and remains uncharted territory in many other clinical areas.  

Dr. med. Ulrich Salzer
University of Freiburg Medical Centre
Centre for Chronic Immunodeficiency (CCI)
Engesserstr. 4
79108 Freiburg
Tel.: +49 (0)761/ 270 - 78 140
Fax: +49 (0)761/ 270 - 78 160
E-mail: ulrich.salzer(at)uniklinik-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/small-rnas-and-immunological-reactions