Intralesional gene expression profile of JAK-STAT signaling pathway and associated cytokines in Leishmania tropica- infected patients

Abstract

Background: The JAK-STAT signaling pathway is a central cascade of signal transduction for the myriad of cytokines in which dysregulation has been implicated in progression of inflammatory and infectious diseases. However, the involvement of this pathway in human cutaneous leishmaniasis (CL) due to Leishmania (L.) tropica warrants further investigation.

Methods: This study sought to investigate differential gene expression of several cytokines and their associated jak-stat genes in the lesions of L. tropica-infected patients byquantitative Real-Time PCR. Further, the expression of five inhibitory immune checkpoint genes was evaluated.

Results: Results showed that the gene expression levelsof both Th1 (ifng, il12, il23) and Th2 (il4, il10) types cytokines were increased in the lesion of studied patients. Further, elevated expression levels of il35, il21, il27 and il24 genes were detected in the lesions of CL patients. Notably, the expression of the majority of genes involved in JAK/STAT signaling pathway as well as checkpoint genes including pdl1, ctla4 and their corresponding receptors was increased.

Conclusion: Our finding revealed dysregulation of cytokines and related jak-stat genes in the lesion of CL patients. These results highlight the need for further exploration of the functional importance of these genes in the pathogenesis of, and immunity to, CL.

Keywords: JAK-STAT signaling pathway; Leishmania tropica; cutaneous leishmaniasis; cytokines; immune checkpoints.

Figure 1

Differential expression gene analysis. (A) Principal Component Analysis (PCA) for skin lesions of CL patients (blue circles, n = 17) compared to the healthy group (red circles, n = 10). (B) Volcano plots of the tested genes in CL patients when compared to a healthy group. (C) Statistically significant up-and down-regulated genes in CL lesions compared to healthy skin. CL, cutaneous leishmaniasis; FC, fold change.

Figure 2

The effect of L. tropica infection on mRNA expression of 11 cytokine genes in the skin lesion of CL patients. Relative mRNA expression of (A) il12a/p35, (B) il12b/p40, (C) il23a/p19, (D) il27b/p28, (E) il27b/ebi3, (F) ifng, (G) ifngr1, (H) ifngr2, (I) il4, (J) il13, (K) il10, (L) il24, (M) il21, and (N) il21r. Data are normalized using gapdh as the control gene. **P < 0.01, and ***P< 0.001. The results were shown as the mean +/- SD of duplicate measurements. CL, Cutaneous leishmaniasis. ns, non-significant.

Figure 3

The expression levels of 10 jak-stats genes and two Th1/2 related-TF between L. tropica infected patients and healthy group. (A) stat1, (B) stat2, (C) stat3, (D) stat4, (E) stat5, (F) stat6, (G) jak1, (H) jak2, (I) jak3, (J) tyk2, (K) tbet, and (L) gata3. Data are normalized using gapdh as the control gene. *P<0.05, **P < 0.01, and ***P< 0.001. The results were shown as the mean +/- SD of duplicate measurements. CL, Cutaneous leishmaniasis.

Figure 4

The expression levels of immune checkpoint genes between L. tropica infected patients and healthy group. (A) pd1, (B) pdl1, (C) ctla4, (D) cd80, (E) cd86. Data are normalized using gapdh as the control gene. ***P< 0.001. The results were shown as the mean +/- SD of duplicate measurements. CL, Cutaneous leishmaniasis.

Figure 5

The relative expression of several genes induced during L. tropica infection. (A) gbp4, (B) gbp5, (C) hif1a, (D) timp4, (E) mmp9, (F) sirpg, and (G) flg. Data are normalized using gapdh as the control gene. ***P< 0.001. The results were shown as the mean +/- SD of duplicate measurements CL, Cutaneous leishmaniasis.

Figure 6

The correlation matrix shows the relationships between cytokines and jak-stat genes in L. tropica-infected patients. The correlation values are displayed in matrices and color-coded: blue for positive correlations and red for negative correlations. Any correlation values that are not statistically significant (P>0.05) are represented by blank white spaces.

Figure 7

Schematic representation illustrating the expression pattern of the genes analyzed within the lesion of CL patients infected with L. tropica. This figure provides a whole overview of the signaling cascade without linking the effect of the cytokines to a specific immune cell. Upon Leishmania entry into the dermis, different innate cells such as neutrophils, macrophages, and dendritic cells infiltrate the site of infection. The cell produces effector molecules like cytokine resulting in either a permissive or hostile environment for the parasite. The effector function of the immune cell also instructs T helper (e.g., tbet/gata3) cell differentiation. In skin resident cells as well as migratory immune cells, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway can be induced by a myriad of cytokines present during skin inflammation. Cytokines (il24, il4, il10, ifng, il12, il27, il35 and il21) interact with the corresponding receptors (ifngr2, and il21r), which phosphorylate JAKs (jak1, jak2, jak3 and tyk2) and activates stat genes (stat1-6). Activated STAT forms a dimer. The dimerized STAT translocate to the nucleus and mediates the transcriptional regulation of target genes like interferon-stimulated gene (ISG; gbp4, and gbp5), and inflammatory molecules. During an immune response, different stimulatory and inhibitory immune checkpoints (pd1/pdl1, ctl4/cd80/cd86) pathways can also induce or suppress. This interaction mainly occurs between a T cell and a macrophage, and DC as antigen-presenting cells (APCs). Up- and down-regulated genes were presented in red and blue colors, respectively. Gene with an unchanged expression was also shown in black. CL, cutaneous leishmaniasis. This image was created with BioRender.com.