Leishmania-encoded orthologs of macrophage migration inhibitory factor regulate host immunity to promote parasite persistence

Abstract

Leishmania major encodes 2 orthologs of the cytokine macrophage migration inhibitory factor (MIF), whose functions in parasite growth or in the host-parasite interaction are unknown. To determine the importance of Leishmania-encoded MIF, both LmMIF genes were removed to produce an mif(-/-) strain of L. major This mutant strain replicated normally in vitro but had a 2-fold increased susceptibility to clearance by macrophages. Mice infected with mif(-/-) L. major, when compared to the wild-type strain, also showed a 3-fold reduction in parasite burden. Microarray and functional analyses revealed a reduced ability of mif(-/-) L. major to activate antigen-presenting cells, resulting in a 2-fold reduction in T-cell priming. In addition, there was a reduction in inflammation and effector CD4 T-cell formation in mif(-/-) L. major-infected mice when compared to mice infected with wild-type L. major Notably, effector CD4 T cells that developed during infection with mif(-/-) L. major demonstrated statistically significant differences in markers of functional exhaustion, including increased expression of IFN-γ and IL-7R, reduced expression of programmed death-1, and decreased apoptosis. These data support a role for LmMIF in promoting parasite persistence by manipulating the host response to increase the exhaustion and depletion of protective CD4 T cells.-Holowka, T., Castilho, T. M., Baeza Garcia, A., Sun, T., McMahon-Pratt, D., Bucala, R. Leishmania-encoded orthologs of macrophage migration inhibitory factor regulate host immunity to promote parasite persistence.

Keywords: CD4 T cells; MIF; apoptosis; exhaustion; inflammation.

Figure 1.

Generation of mif^−/− L. major. A) Separate plasmid-targeting constructs were generated with regions upstream of Lm1740MIF and downstream of Lm1750MIF flanking resistance cassettes to hygromycin or puromycin. For genetic reconstitution, a 1.1 kb region of genomic DNA encompassing both Lm1740MIF and Lm1750MIF was cloned into the pXG expression vector and stably transfected into the mif^−/− strain, and mif^−/−TG parasites resistant to G418 were selected. B) PCR performed on genomic DNA of wild-type, mif^−/−, or mif^−/−TG parasites with primers directed to Lm1740MIF or Lm1750MIF. A third PCR with primers to amplify an upstream sequence of Lm1740MIF and within the hygromycin resistance cassette verified proper insertion of the targeting construct. C) qPCR performed on wild-type, mif^−/−, and mif^−/−TG parasite genomic DNA or cDNA with primers targeted to amplify Lm1740MIF and Lm1750MIF. D) Sandwich ELISA performed to detect Lm1740MIF in lysates or culture medium from wild-type, mif^−/−, and mif^−/−TG promastigote-stage parasites grown to log phase (3–4 d). C, D) Each experiment was performed 3–4 times (n = 4 for each parasite strain). *P < 0.05; ***P < 0.005.

Figure 2.

Fitness of mif^−/− L. major and persistence in host macrophages. A) Wild-type, mif^−/−, and mif^−/−TG parasites were grown in culture medium without antibiotics, and the number of parasites was determined with a hemocytometer. The experiment was performed 3 times (n = 5 for each parasite strain). B) BALB/c macrophages were infected with wild-type, mif^−/− , or mif^−/−TG L. major at an MOI of 5 and fixed and labeled with DAPI, and the parasites were recorded by enumerating by parasite nuclei vs. macrophage nuclei. The experiment was performed 3 times (n = 4 for each parasite strain). C) Wild-type macrophages stimulated with LPS 4 h after infection followed by counting of DAPI-labeled parasite nuclei in fixed cells. Each experiment was performed 5 times (n = 3–4 for each parasite strain used). ***P < 0.005 for mif^−/− vs. wild-type and mif^−/− vs. mif^−/−TG. D) BALB/c macrophages were stimulated with LPS 4 h after infection with wild-type, mif^−/−, or mif^−/−TG parasites, and nitrite content was measured in the culture medium at 48 h. The experiment was performed 3 times (n = 4 for each condition). E) Histograms of log₁₀ fluorescence of TUNEL staining in wild-type macrophages infected with wild-type, mif^−/−, or mif^−/−TG parasites 48 h after stimulation with LPS. Percentage of TUNEL⁺ macrophages determined using flow cytometry. Each experiment was performed 4 times (n = 3–4 for each condition). *P < 0.05, **P < 0.01. F) Cd74^−/− macrophages infected with wild-type, mif^−/−, or mif^−/−TG parasites 48 h after stimulation with LPS. The percentage of TUNEL⁺ macrophages determined using flow cytometry. Each experiment performed 4 times (n = 3–4 for each condition). G) Cd74^−/− macrophages stimulated with LPS 4 h after infection followed by counting of DAPI-labeled parasite nuclei in fixed cells. Each experiment was performed 5 times (n = 3–4 for each parasite strain used).

Figure 3.

Infection and disease progression in mice affected by LmMIF. A) BALB/c mice were infected with 10⁶ wild-type or mif^−/− L. major intradermally in the hind foot, and the lesion size was measured with a microcaliper. B) DNA extracted from lesions of mice infected for 4 or 12 wk and parasite burden determined using qPCR to measure relative quantity of Lm kDNA. Each experiment was performed 3 times (n = 8–10 for each condition). *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 4.

Inflammatory state at the site of infection. A) H&E staining of tissue sections from skin overlying the infected foot of naïve BALB/c mice or BALB/c mice infected for 4 wk with wild-type or mif^−/− L. major. Magnification, ×100 (top row); ×400 (bottom row).. Scale bar, 200 μm (top); 50 μm (bottom). Number of total immune cells (including myeloid cells and lymphocytes) counted per ×400 field. The experiment was performed 3 times (n = 3–4 for each condition). *P < 0.05. B) Cells were collected from infected lesion tissue and enumerated, and the phenotype was determined by flow cytometry gated for CD3⁺ T lymphocytes, CD11b^HiGR1^Hi neutrophils, CD11b⁺Ly6C^Hi monocytes, CD11b⁺F4/80^Hi macrophages, and CD11b⁺CD11c^HiMHC II^Hi DCs. The experiment was performed 3 times (n = 3–4 for each condition). ^#P < 0.1; *P < 0.05. C) RNA extracted from lesions 4 wk after infection with 10⁶ parasites and subjected to qPCR to determine gene expression. Represented as fold expression relative to that of mice infected with wild-type L. major. The experiment was performed 4 times (n = 3–5 for each gene analyzed). *P < 0.05; ***P < 0.005.

Figure 5.

Whole-genome microarray expression analysis of infected macrophages. A) Venn diagrams of number of upregulated or downregulated by >1.5-fold in BALB/c macrophages infected with wild-type L. major vs. those infected with mif^−/− L. major, as determined by microarray analysis. P < 0.05. B) A heat map depicting relative gene expression in naïve macrophages or macrophages infected with wild-type or mif^−/− L. major for 4 or 16 h. Genes from cellular pathways of interest are displayed. Each column represents a separate replicate (n = 3 for each). C) qPCR of cDNA from macrophages infected with wild-type or mif^−/− L. major for 16 h with primers directed to amplify macrophage genes, or Leishmania kDNA. Displayed as expression relative to wild-type infected macrophages. Experiment performed 3 times (n = 3–4 for each gene analyzed). *P < 0.05; **P < 0.01.

Figure 6.

Phenotype and activity of APCs impacted by LmMIF. A) Wild-type or (C) Cd74^−/− BALB/c macrophages and DCs infected with wild-type, mif^−/− or mif^−/−TG L. major (MOI = 10) were analyzed after 24 h for surface marker expression by flow cytometry. Macrophages were gated on CD11b⁺ cells and DCs on CD11c⁺ cells. Each set of plots is representative of 4 separate experiments. B) Wild-type or (D) Cd74^−/− macrophages or DCs infected for 4 h were cocultured with LMR7.5 T cells, and media were collected after 24 or 48 h for IL-2 measurement by ELISA. Each experiment was performed 3 times (n = 5 for each condition). ^#P < 0.1; ***P < 0.001.

Figure 7.

Effector T_H cell formation affected by LmMIF. A) qPCR performed on cDNA from draining lymph node cells and expression of genes quantified and represented as fold expression relative to that of mice infected with wild-type L. major. The experiment was performed 3 times (n = 5–6 for each gene analyzed). B) Cells collected from draining popliteal lymph nodes of mice infected for 4 wk with wild-type or mif^−/− L. major analyzed with flow cytometry. Cells gated on CD4⁺ T_H cells and log₁₀ fluorescence of CD44 and intranuclear Ki67 expression or (C) CD62L expression was observed. Mean fluorescence intensity of CD44 and frequency of Ki67⁺ cells were quantified. D) Lymph node cells from mice infected with wild-type or mif^−/− L. major were cultured with soluble Leishmania antigen (SLA) for 48 h and cytokine production determined by ELISA. Experiment performed 3 times (n = 5 for each condition). E) CD4⁺Ki67⁺CD44hi cells observed with flow cytometry for log₁₀ fluorescence of IFN-γ and the frequency of IFN-γ⁺ cells quantified. D, E) Experiments were performed 3 times (n = 6–8 for each condition). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8.

Increased exhaustion and reduced viability of effector T_H cells. A–C) CD4⁺Ki67⁺CD44^hi T cells observed with flow cytometry for log₁₀ fluorescence of expression of PD-1, IFN-γ (A) , and IL-7R (B). The frequency of PD-1⁺ and IL-7R^hi cells was quantified. C) qPCR was performed on cDNA from lymph node cells, and the expression of IL-7R and HPRT was quantified relative to Hprt and represented as fold expression relative to that of mice infected with wild-type L. major. Experiment performed 3 times (n = 3–5 for each gene analyzed). *P < 0.05. D) CD4⁺CD44^hi cells (IL7R^hi, IL7R^lo or total) were observed with flow cytometry for log₁₀ fluorescence of TUNEL staining, and the frequency of TUNEL⁺ cells was quantified. A, B, D) Experiments were performed 3 times (n = 6–8 for each condition). *P < 0.05; **P < 0.01; ***P < 0.005.

Figure 9.

Effector T_H-cell response to challenge infection. A) CD4 T cells purified from the draining lymph nodes of BALB/c mice 4 wk after infection with 10⁶ wild-type or mif^−/− L. major were transferred into SCID mice that were subsequently infected with wild-type L. major. Two weeks later, splenocytes were collected from SCID mice, and CD4⁺ CD44^hi cells were observed with flow cytometry for log₁₀ fluorescence of CFSE labeling and IFN-γ expression. Experiment performed 3 times (n = 5–6 for each condition). **P < 0.01; ***P < 0.005. B) BALB/c mice infected for 4 wk with 10⁶ wild-type or mif^−/− L. major in the right foot, then challenged with 5 × 10⁶ wild-type parasites in the left foot. Draining lymph node CD4⁺ T_H cells were observed for expression of Ki67 and IFN-γ.The frequency of IFN-γ and Ki67 expression on the CD4⁺ cells was quantified. The experiment was performed 3 times (n = 4 for each condition). *P < 0.05. C) Cells from draining lymph nodes at the site of the secondary challenge were cultured 48 h with SLA, and cytokine production was determined by ELISA. Experiment performed 3 times (n = 4 for each condition). N.S., no significance. *P < 0.05.

Figure 10.

Summary of the overall impact of LmMIF during Leishmania infection. LmMIF is released by L. major and signals through the MIF receptor (CD74) on host macrophages to block apoptosis and prevent clearance of internalized parasites. Genes related to immune activation and inflammation are also upregulated by LmMIF, leading to enhanced antigen presentation by DCs. Effector T-cell priming and proliferation are accelerated; however these cells are nonfunctional and more readily succumb to exhaustion and apoptosis. The formation of long-lived effector T_h cells is consequently inhibited, resulting in impaired control of chronic infection with L. major.