NOD Like Receptors (NLRs) in Human B Cells

We all know that innate immune response provides first line of defense against invading micro-organisms. Cells of the innate immune system express certain proteins that are called pattern recognition receptors (PRRs). These receptors identify microbe associated molecular patterns (MAMP) or pathogen associated molecular patterns (PAMP), which are associated with microbial pathogenesis. These PRRs can be divided into two types based on their presence on/in the cells. These two types include membrane bound PRRs (most famous example is Toll like rector (TLR), and cytoplasmic PRRs.

The NOD like receptors (NLRs) are the cytoplasmic PRRs. The NLR family comprise of 20 intracellular proteins that recognize primarily bacterial structures. They are divided into three subfamilies, NODs, NALPs and NAIPs. The best characterized members of the NOD sub-family are NOD1 and NOD2. NOD1 recognizes a molecule called meso-DAP, specific for Gram negative bacteria and NOD2 recognizes MDP, a component of all types of bacterial peptidoglycans. In the NALP subfamily, NALP1beta and NALP3 are most well-known members. Members of NALP family respond to danger signals released from injured or dying cells, leading to formation of inflammasomes. The members of NAIP family detect intracellular flagellin and also lead to the formation of inflammasome. Expression of NLRs has been detected in many cells including DCs, macrophages, monocytes, epithelial and endothelial cells and osteoblasts. However, at present it is not known whether B cells also express NLRs or not. In the present study, the authors thus investigated the expression of various NLRs and their function in human peripheral and tonsillar B cell subsets. The authors also explored the cooperative roles of NLRs and selected TLRs in B cell activation.

This paper is from the laboratory of Dr. Lars-Olaf Cardell, Division of ENT Diseases, CLINTEC, Karolinska Institute, Sweden and is published in Journal of leukocyte Biology, September 2010 issue.

Petterson T, Jendholm J, Månsson A, Bjartell A, Riesbeck K, Cardell LO. Effects of NOD-like receptors in human B lymphocytes and crosstalk between NOD1/NOD2 and Toll-like receptors. J Leukoc Biol. 2010 Sep 15.

 

A summary of major results obtained by the investigators is as follows:

• Expression of NLRs in human B lymphocytes: Freshly isolated peripheral and tonsillar and B lymphocytes (from patients) were used and mRNA expression of NOD1, NOD2, NALP1, NALP3, NAIP, and IPAF was analyzed using real-time PCR. The results of the PCR assay showed that peripheral B cells expressed all the NLRs tested in this experiment, except IPAF. In contrast, tonsillar cells only expressed NOD1, NALP1, and NAIP. Furthermore, flow cytometric and immunohistochemistry analyses were done to confirm that results of real-time PCR. For these analyses, rabbit polyclonal Ab against NOD1 and NAIP and mouse mAb against NOD2, NALP1 and NALP3 were used. It was observed that peripheral B cells expressed all the tested receptors, while tonsillar B cells showed expression of NOD1, NALP1 and NAIP.
  
• B cell receptor triggering is required for NLR-induced B cell activation: To determine whether NLRs have the capability to induce B cell activation upon stimulation with their ligands, purified B cells were isolated and cultured with or without iE-DAP (NOD1 ligand) and MDP (NOD2 ligand) in the presence or absence of BCR i.e., alpha IgM or IgD-binding protein MID. It was observed that when B cells were cultured with NOD1/2 ligands only, no proliferation was observed. However, when cells were stimulated with NLR-ligands in combination with BCR cross-linking via IgM or IgD, a profound increase in B cell proliferation was observed. To examine the role of physical T cell help in NOD induced B cell proliferation, the authors added rCD40L to highly purified B cells. They observed that CD40L in combination with the NLR ligands did not lead any additional proliferation of B cells. To find out more about NLR-induce B cell activation, the investigators stimulated B cells with NOD1/2 ligands together with BCR cross-linking (IgM/IgD) and analyzed expression of cell surface glycoproteins and viability. The number of CD27+, CD69+, CD71+, CD80+, CD86+, and CD95+ B cells was found to be increased. In peripheral B cells, the expression of all these markers were found to be elevated in comparison to unstimulated cells or to those stimulated with BCR alone. In tonsillar B cells, only stimulation with NOD1 ligand increased the percentage of CD71+, CD80+, CD86+, and CD95+ cells. It was also observed that viability of B cells cultured with NOD1 ligands along with BCR was slightly enhanced than controls. However, culture with NOD2 ligands along with BCR did not cause any increase in viability. As tonsillar B cells showed lower B cell responses, the next question was why? To answer this question, investigators determined the level of NLR proteins in naïve (CD19+IgD+CD38-), germinal center (CD19+IgD-CD38+) and memory (CD19+IgD-CD38-) B cells. However, they could not find any difference between blood derived and tonsillar cells.
  
• NLR stimulation augments TLR-induced B cell proliferation: To study the interplay between NLRs and TLRs on B cells, the investigators incubated B cells with suboptimal concentration of TLR agonists in combination with NOD1/2 ligands and BCR cross-linking. It was observed that proliferation of peripheral B cells was enhanced, when they were incubated with TLR agonists. In contrast, only co-stimulation with NOD1 ligand increased the proliferation in tonsillar B cells.
  

Conclusions:

1. This is the first study to demonstrate the presence of a range of NLRs in purified human B cells.
   
2. The study shows that peripheral B cells express NOD1, NOD2, NALP1, NALP3 and NAIP, while tonsillar B cells express NOD1, NALP1 and NAIP.
   
3. Activation of B cells by NLR ligands required BCR cross linking and did not require T cell help.
   
4. NLRs enhanced TLR-induced activation of B cells.