Leishmania donovani infection induces anemia in hamsters by differentially altering erythropoiesis in bone marrow and spleen.

Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (α-globin, β-globin, ALAS2) were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR) and transcription factors (GATA1, GATA2, FOG1) were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-γ mRNA is highly increased by L. donovani infection. Expression of the IFN-γ inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL), was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by cytokine-mediated apoptosis of erythroblasts

<i>L. donovani</i> infected hamsters develop severe anemia and leukopenia.

<p>Hematological analysis of blood from control hamsters and hamsters infected with <i>L. donovani</i> for 8 weeks. Data shown are from individual hamsters and the mean. Hemogloblin levels (A) and hematocrit (B) were statistically significant at p<0.01. Red blood cell (C) and white blood cell counts (D) were significant at p<0.05, Student’s t test.</p

List of primers used for RT-PCR.

<p>List of primers used for RT-PCR.</p

<i>L. donovani</i> infection increases BFU-E and CFU-E progenitor numbers in the bone marrow and spleen.

<p>(A) Frequency of BFU-E progenitors in the bone marrow of control hamsters and hamsters infected with <i>L. donovani</i> for 8 weeks was determined by the semi-solid colony assay. Data represent mean +/− SEM from hamsters. *p<0.05, Student’s t test. (B) and (C) Frequency of CFU-E progenitors in bone marrow and spleen from control and infected hamsters. Data represent mean +/− SEM from 3–5 hamsters. *p<0.05; *p<0.01, student’s t test.</p

T cell and anti-inflammatory cytokine mRNA are increased by <i>L. donovani</i> infection.

<p>mRNA levels of T cell cytokines IL-2 and IL-4 (A and B) and anti- inflammatory cytokines IL-10 and TGF-β (C and D) in the spleen and bone marrow of control non-infected hamsters and <i>L. donovani</i> infected hamsters were determined by real-time RT-PCR, mRNA levels are expressed relative to control non-infected hamsters. Data represent mean +/− SEM of 6–8 hamsters in each group. *p<0.05; **p<0.01; ***p<.001, Student’s t test.</p

<i>L. donovani</i> infection increases apoptosis of erythroblasts.

<p>Apoptosis of bone marrow erythroblasts was examined by flow cytometry from control non-infected hamsters and <i>L. donovani</i> infected hamsters. Apoptosis was determined by Annexin-V binding (A) and the TUNEL Assay (B). Representative flow cytometry plots are from a control non-infected hamster and a hamster infected with <i>L. donovani</i> for 7 weeks. (A) Bone marrow cells were labeled with anti- E-cadherin antibody-e-Fluro 660 and FITC-Annexin V. Dead cells were excluded with 7-amino actinomycin (7-ADD). (B) BrdUTP incorporation by the TUNEL technique in erythroblasts was detected using Alexa-488 anti-BrdUTP and anti-E-cadherin-e-Fluro 660. (C) Percentage of E-cadherin positive erythroblasts that are Annexin-V positive. (D) Percentage of E-cadherin positive erythroblasts that are TUNEL positive. Data represent mean +/− SEM of 4 hamsters in each group. *p<0.05, Student’s t test.</p

Erythropoietin and Iron levels in infected hamsters.

<p>(A) Serum erythropoietin (Epo) levels in control hamsters and hamsters infected with <i>L. donovani</i> for 5 and 8 were determined by ELISA. EPO levels were significantly increased by infection, p<0.005, one-way ANOVA. (B) Serum iron levels in control and infected hamsters were determined by ferrozone assay. Serum iron levels were significantly decreased by infection, p<0.0005, one-way ANOVA. (C). Liver iron levels in control and infected hamsters were determined by ferrozone assay. Liver iron levels were significantly increased by infection p<0.05, one-way ANOVA.</p

IFN-γ and TRAIL mRNA are highly expressed in <i>L. Donovani</i> infected hamsters.

<p>mRNA levels of IFN-γ (A) and TNF-related apoptosis inducing ligand (TRAIL) (B) were determined by real-time RT-PCR in the bone marrow and spleen of control non-infected hamsters and <i>L. donovani</i> infected hamsters. mRNA levels are expressed relative to control non-infected hamsters. Data represent mean +/−SEM of 6–8 hamsters in each group. **p<0.01; ***p<0.001, Student’s t test.</p

Erythroid gene expression is differentially altered by <i>L. donovani</i> infection in the bone marrow and spleen.

<p>mRNA levels of erythroid genes were determined by real-time RT-PCR in bone marrow (A,C,E) and spleen (B, D, F). mRNA levels were normalized to β-actin and expressed relative to control non-infected hamsters. Data represent mean +/− SEM of 6–8 hamsters in each group. *p<0.05; **p<0.01, Student’s t test.</p