Integrated pathways for neutrophil recruitment and inflammation in leprosy

Abstract

Neutrophil recruitment is pivotal to the host defense against microbial infection, but it also contributes to the immunopathology of disease. We investigated the mechanism of neutrophil recruitment in human infectious disease by means of bioinformatic pathways analysis of the gene expression profiles in the skin lesions of leprosy. In erythema nodosum leprosum (ENL), which occurs in patients with lepromatous leprosy and is characterized by neutrophil infiltration in lesions, the most overrepresented biological functional group was cell movement, including E-selectin, which was coordinately regulated with interleukin 1beta (IL-1beta). In vitro activation of Toll-like receptor 2 (TLR2), up-regulated in ENL lesions, triggered induction of IL-1beta, which together with interferon gamma induced E-selectin expression on and neutrophil adhesion to endothelial cells. Thalidomide, an effective treatment for ENL, inhibited this neutrophil recruitment pathway. The gene expression profile of ENL lesions comprised an integrated pathway of TLR2 and Fc receptor activation, neutrophil migration, and inflammation, providing insight into mechanisms of neutrophil recruitment in human infectious disease.

Figure 1

(A) Unsupervised data analysis separates leprosy lesion samples into clinically relevant subclasses based on their gene expression patterns. Hierarchical clustering analysis divides 7 L-lep and 6 ENL skin lesion samples into two distinct groups that cluster on separate branches of a dendrogram. There are 3158 probe sets represented in this diagram. (B) Permutation analysis of the microarray data reveals that only less than 0.1% of the permutated groupings manifest more distinction in gene expression than the defined ENL / L-lep patient grouping. The cumulative numbers of probests (Y-axis) with Student’s t-test p-values less than various threshold levels (X-axis) were calculated for the clinically relevant ENL / L-lep grouping and plotted (black). One thousand randomly permutated groupings were also generated and tested. We plotted the mean (red), 10% (green), 1% (blue), and 0.1% (yellow) number of probesets below a given p-value among the permutated groupings and compared these to the correct ENL / L-lep grouping. Compared to the 0.1% confidence level, the ENL / L-lep grouping generally has more differentially expressed probesets with p-values below the indicated threshold, indicating that the ENL / L-lep grouping is statistically significant. (C) Prediction accuracy using leave-one-out cross-validation and weighted gene-voting. Using the ENL / L-lep grouping, our prediction algorithm correctly assigned the subclasses of 12 out of 13 samples with high confidence (prediction strength >0.4).

Figure 2

Enriched pathways in ENL vs. L-lep. The functional and canonical pathways analyses were generated through the use of Ingenuity Pathways Analysis (www.ingenuity.com). (A) The top 20 biological functions in ENL vs. L-lep are ranked by their p-values (x-axis). (B) All statistically significant canonical pathways represented by the differentially expressed genes are shown ranked by their p-values (x-axis). Fischer’s exact test was used to calculate a p-value determining the probability that each pathway represented by the expression data is due to chance alone.

Figure 3

(A) Genes within the top functional group represented in ENL skin lesions, cell movement, also belonging to a subset involved with neutrophil function. Each row represents the gene expression profile of a particular probe corresponding to the gene listed, individual squares represent the relative gene expression intensity of the given gene in a patient with red being relatively increased in expression and green relatively decreased in expression. (B) Diagram illustrating the cellular location of each of the genes in (A). Cell type-specific expression (neutrophil-blue, endothelial cell-pink, both-purple or neither-gray) is shown for those proteins whose cellular location is in the plasma membrane.

Figure 4

(A) Correlation of E-selectin expression with IL-1β and IL-1R1. Shown are the R² values (Pearson's correlation) between the expression of E-selectin and the expression of IL-1β and IL-1R1. (B) E-selectin and von Willebrand factor protein expression in L-lep compared to ENL skin lesions in vivo. Thin (4 µm) sections of leprosy biopsy samples were incubated with anti-E-selectin or VWF and stained secondarily with an immunoperoxidase method followed by counterstaining with hematoxylin. Each bar denotes 100 µm. The isotype controls were negative. The findings shown are representative of five patients in each group.

Figure 5

Effect of FcR or TLR2/1 activation of peripheral blood monocytes on IL-1β (A) and IL-8 (B) secretion. Percolled monocytes from healthy donors were stimulated overnight with a lipopetide known to stimulate TLR2/1 or plate-bound hIgG in media (open squares), thalidomide R (filled triangles) or thalidomide S (filled circles) enantiomers. IL-1β and IL-8 were measured from cultured supernatant by ELISA. Shown are averages of triplicate cultures +/− SEM. Asterisks denote differences between media and thalidomide-treated cells with p<0.05 calculated by paired Student’s t-test.

Figure 6

(A) Effect of IFN-γ on IL-1β induction of E-selectin expression on HUVECs. (B) Effect of thalidomide on E-selectin and ICAM-1 expression of HUVECs induced by IL-1β and IFN-γ. (C) Neutrophil binding to HUVEC cells stimulated with IL-1β and IFN-γ in the presence or absence of thalidomide. Data shown are a compilation of 3 independent experiments. Statistical analysis was performed by paired Student’s t-test.

Figure 7

A gene program of neutrophil recruitment and tissue injury based on gene expression and in vitro data. Molecules in black text represent those added to the pathway based on microarray data, while molecules/genes in blue text have supportive inmmunohistochemical and/or in vitro data. Immune complexes and/or mycobacterial components may activate FcRs and TLRs to induce pro-inflammatory cytokines such as IL-8 and IL-1β. IL-1β in combination with IFN-γ induce E-selectin expression on endothelial cells, resulting in the first step of neutrophil recruitment, adhesion and rolling along the endothelial wall. These steps are inhibited by thalidomide. Neutrophil interaction with ICAM-1 as well as other integrin-mediated activators results in arrest and activation. Glycosaminoglycans such as heparan sulfate and extracellular proteins such as plasminogen activator inhibitor 1 promote the presentation of chemokines such as IL-8 on the surface of the endothelium to mediate neutrophil activation and chemotaxis, inhibited by secretory component. As part of the innate immune response to microbial infection, the local recruitment of neutrophils may lead to tissue injury as host defense pathways are executed.