Transcriptional Profiling of Immune-Related Genes in Leishmania infantum-Infected Mice: Identification of Potential Biomarkers of Infection and Progression of Disease

Abstract

Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. In this large-scale analysis, differential expression of 112 immune-related genes has been analyzed using high-throughput qPCR in spleens of infected and naïve Balb/c mice at four different time points. This analysis revealed that early response against Leishmania infection is characterized by the upregulation of Th1 markers and M1-macrophage activation molecules such as Ifng, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3. This activation doesn't protect spleen from infection, since parasitic burden rises along time. This marked difference in gene expression between infected and control mice disappears during intermediate stages of infection, probably related to the strong anti-inflammatory and immunosuppresory signals that are activated early upon infection (Ctla4) or remain activated throughout the experiment (Il18bp). The overexpression of these Th1/M1 markers is restored later in the chronic phase (8 wpi), suggesting the generation of a classical “protective response” against leishmaniasis. Nonetheless, the parasitic burden rockets at this timepoint. This apparent contradiction can be explained by the generation of a regulatory immune response characterized by overexpression of Ifng, Tnfa, Il10, and downregulation Il4 that counteracts the Th1/M1 response. This large pool of data was also used to identify potential biomarkers of infection and parasitic burden in spleen, on the bases of two different regression models. Given the results, gene expression signature analysis appears as a useful tool to identify mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development.

Anti-Leishmanial Vaccines: Assumptions, Approaches, and Annulments

“…Of the 112 genes studied, 22 showed differential expression in infected vs. uninfected mice. Il12rb2, Il23r, and Ptgs2 were exclusively expressed only in infected mice but not in the control group, suggesting that the immune molecules may vary drastically under the condition when initial priming is done (uninfected) compared to when subsequent infection occurs [190]. Thus, an aim in vaccination is to induce the balanced expression of co-stimulatory and co-inhibitory molecules such that appropriate Th cell priming occurs.…”

Macrophage Polarization in Leishmaniasis: Broadening Horizons

“…This controversy may be explained by the fact that different patterns of gene expression are found in macrophages at different time points ( 130 ). A recent study demonstrated that the early response against L. donovani infection was distinguished by the increase in of Th1 markers and M1-macrophage activation molecules (IFN-γ, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3).…”

“…However, this activation was not protective because the parasitic burden increased over time. There was no marked overlap of macrophage phenotypes at intermediate times of infection, and the overexpression of these Th1/M1 markers was restored later in the chronic phase without parasitic burden control ( 130 ).…”

Integrated multivariate analysis of transcriptomic data reveals immunological mechanisms in mice afterLeishmania infantuminfection

“…Statistically significant differences are indicated (*p < 0.05; **p < 0.01, ***p < 0.001). Global analysis of transcriptome generated under different experimental conditions, has grown increasingly relevant in order to clarify key questions of intricate models like infection of target organs in leishmaniasis (11,13). With the aim of deciphering the most relevant aspects of the response against leishmaniasis developed in liver during the initial phases of infection with L. infantum, a massive real-time quantitative PCR analysis (more than 32000 qPCR reactions) was performed in BALB/c mice.…”

“…Many studies have focused on the analysis of immunological response in the spleen in Leishmania-infected mice, (11)(12)(13), where many mediators play a role in M1/Th1 and M2/Th2 responses, but also markers of T reg response, Th17, and Tfh cells are involved (14). However, understanding the development of immunity in the liver, where infection develops shortly after inoculation, could lead to potential strategies to improve the elimination of the parasite in the spleen (15).…”