A study on Leukocyte Ig like receptors in the context of infection

Leukocyte Ig like receptors (LILR or ILT or ILR) are a family of innate immune receptors. These are predominantly expressed on the cells of the myelomonocytic lineage. This family includes 11 proteins that can be further subdivided into activating (subfamily A) or inhibitory (subfamily B) receptors on the basis of their transmembrane or cytoplasmic domains. A lot of research has been going on their role in immune dysregulation and following transplantation. However, not much is known on their role during infection. LILBR4 and LILBR2 are two such important receptors. They have the potential to inhibit T cell proliferation and thus they have been extensively studied in the context of allograft tolerance in case of transplantation and in cancer. The aim of the present work was to better understand the inhibitory potential of LILBR2 and LILBR4 during infection. Salmonella typhimurium is an obligate intracellular pathogen that infects macrophages. In the present study, the modulation of expression of these receptors on antigen presenting cells infected with S. typhimurium was determined.

Cell surface phenotype of LILRB4 ligated dendritic cells= To study this, dendritic cells were cultured in the presence of an isotype control antibody (control), LILBR4 specific antibody with protein G. LPS matured dendritic cells were also cultured in the presence of an isotype control antibody (control) and LILBR4 specific antibody. On these cells flow cytometric analysis of cell surface markers (CD80, CD86, CD83, CD1a) was performed. Ligation of LILBR4 led to an upregulation of costimulatory molecule CD86 on both immature and LPS matured dendritic cells. No significant effects on expression of CD83, CD80 and Cd1a was observed.

To test the effect of ligation of LILBR4 on dendritic cells on T cell stimulation, the immatured and matured dendridic cells were cultured in the presence of isotype control and LILBR4 specific antibody as before. To these cells naive CD4 cells from an allogenic donor were added in various stimulator: responder ratios and cultured for 6 days. On day 6 of culture, triatiated thymidine was added, cells were incubated and analysed using a scintillation plate counter. These experiments are called mixed leukocyte reactions (MLR). The results of such five MLR experiments showed no increase or decrease in T cell proliferation in response to ligation of LILBR4 on dendritic cells.

To study the effect of ligation of LILBR4 on cytokine secretion profile of macrophages, these cells were cultured in the presence of isotype control, LILBR4 specific antibody. Same was done for LPS matured APCs. The concentration of IL-8 appeared to decrease while concentration of IL-10 increased by ligation of LILBR4 in both mature and immature cells. The results were not significant though.

TTo study the effect of S. typhimurium infection on the expression of LILBR4 and LILBR2- monocyte derived macrophages were infected with S. typhimurium (transformed with pFVP25.1-GFP plasmid). Infection of macrophages was confirmed by GFP expression study by flowcytometry. To study the expression of LILBR2 and LILBR4, RNA was extracted from these macrophages and real time PCR was performed. Similar experiments were done on macrophages infected with heat killed bacteria. An upregulation of LILBR2 and LILBR4 was observed in both the cases.

To study the effect of bacterial components (LPS, TLR4 ligand and flagellin TLR-5 ligand) on LILBR2 and LILBR4 expression, macrophages were treated with S. typhimurium LPS, and flagellin. It was observed that treatment of macrophages with LPS led to an increase in upregulation of both LILBR2 and LILBR4 as measured by real time PCR assay. Upregulation was less pronounced for cells treated with flagellin.

The major conclusions of this study are:
1. Continuous ligation of LILBR4 on dendritic cells leads to increased upregulation of CD86. Previous studies have shown that ligation of LILBR2 results in failure to upregulate CD86.
2. The LILBR4 cross linking did not result in any changes in surface phenotype of APCs that could inhibit T cell proliferation. These results complement previous works which show that a soluble form of LILBR4 is sufficient to inhibit T cell stimulation.
3. The LILBR4 ligation showed an increased production of IL-10 (anti-inflammatory factor) and reduced production of IL-8 a potent inflammatory factor.
4. Infection of macrophages with Salmonella led to an increased expression of LILBR2 and LILBR4. Similar upregulation of expression of LILBR2 and LILBR4 was observed when cells were treated with Salmonella LPS or heat killed Salmonella. These results show that the upregulation was due to LPS.

Future studies that need to be done:
1. To determine whether culture of T cells with LILBR4-crosslinked APCs lead to alterations in T cell functions or alterations in the ration of regulatory and suppressor cells.
2. The effect of LILR and TLR signalling on each others expression and function.