Study of the mechanisms responsible for anemia and thrombocytopenia in an experimental mouse model of visceral leishmaniasis

Visceral leishmaniasis (VL) is a neglected tropical disease caused by protozoan parasites of the Leishmania genus causing between 20 000 and 50 000 deaths per annum. The parasites have developed a range of mechanisms to avoid the host’s immunity and establish chronic infection of the spleen, liver and bone marrow as intracellular parasites of macrophages. The non-exhaustive list of syndromes associated with VL include hepatomegaly, splenomegaly, fever and pancytopenia. The causes for the reduction of red blood cells, platelets or leukocytes are still unclear. Many studies have focused on the immunological aspects of VL, both in humans and experimental models, but the mechanisms causing haematological disorders remain unclear. In this study, it is shown that mice chronically infected with Leishmania donovani (L. donovani) develop haematological abnormalities, namely anaemia and thrombocytopenia. Erythropoiesis was quantified in the bone marrow, the main site of haematopoiesis in adult mammals. The number of late erythroid precursors was severely reduced in infected animals. Reduction of medullar erythropoiesis was associated with a reduction of stromal support in the bone marrow shown by a reduction of stromal macrophages expressing high levels of CD169 and a loss of CXCL12-producing stromal fibroblasts. The granulocyte-colony stimulating factor (G-CSF) known to deplete stromal macrophages and inhibit CXCL12 expression was systematically up-regulated in infected mice. Splenomegaly correlated with compensatory extramedullary erythropoiesis confined to the red pulp. Infection of splenectomised mice demonstrated that anaemia was independent of the spleen since medullar erythropoiesis was still impaired in these mice. Infection caused an increase in CD4 and CD8 T cells in the bone marrow and infected B6 RAG2-/- mice lacking mature T and B cells were not anaemic and had no repression of medullar erythropoiesis nor splenomegaly. Alterations of bone marrow stromal cells or up-regulation of G-CSF did not occur in these mice. Splenomegaly was relevant because it was shown to be responsible for thrombocytopenia. Megakaryopoiesis was unaltered by chronic infection and infected splenectomised mice had higher platelet counts than their sham-operated counterparts. Platelet production could be stimulated by injections of recombinant thrombopoietin (TPO) in chronically infected mice. Efficacy of TPO treatment in curing thrombocytopenia correlated negatively with the severity of splenomegaly. The original contribution of this work is the demonstration of a complex immunopathological mechanism causing haematological changes in an experimental model of VL. Better understanding of haematological alterations of VL is a step forward for the improvement of VL therapy, in which these alterations have been associated with the lethality of the disease

Dissecting pathways to thrombocytopenia in a mouse model of visceral leishmaniasis

Visceral leishmaniasis is an important yet neglected parasitic disease caused by infection with Leishmania donovani or L infantum. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulation, and extensive hematological complications. Thrombocytopenia is a dominant hematological feature seen in both humans and experimental models, but the mechanisms behind this infection-driven thrombocytopenia remain poorly understood. Using a murine model of experimental visceral leishmaniasis (EVL), we demonstrated a progressive decrease in platelets from day 14 after infection, culminating in severe thrombocytopenia by day 28. Plasma thrombopoietin (TPO) levels were reduced in infected mice, at least in part because of the alterations in the liver microenvironment associated with granulomatous inflammation. Bone marrow (BM) megakaryocyte cytoplasmic maturation was significantly reduced. In addition to a production deficit, we identified significant increases in platelet clearance. L donovani-infected splenectomized mice were protected from thrombocytopenia compared with sham operated infected mice and had a greater response to exogenous TPO. Furthermore, infection led to higher levels of platelet opsonization and desialylation, both associated with platelet clearance in spleen and liver, respectively. Critically, these changes could be reversed rapidly by drug treatment to reduce parasite load or by administration of TPO agonists. In summary, our findings demonstrate that the mechanisms underpinning thrombocytopenia in EVL are multifactorial and reversible, with no obvious residual damage to the BM microenvironment

Therapeutic gene editing of T cells to correct CTLA-4 insufficiency

Heterozygous mutations in CTLA-4 result in an inborn error of immunity with an autoimmune and frequently severe clinical phenotype. Autologous T cell gene therapy may offer a cure without the immunological complications of allogeneic hematopoietic stem cell transplantation. Here, we designed a homology-directed repair (HDR) gene editing strategy that inserts the CTLA-4 cDNA into the first intron of the CTLA-4 genomic locus in primary human T cells. This resulted in regulated expression of CTLA-4 in CD4+ T cells, and functional studies demonstrated CD80 and CD86 transendocytosis. Gene editing of T cells isolated from three patients with CTLA-4 insufficiency also restored CTLA-4 protein expression and rescued transendocytosis of CD80 and CD86 in vitro. Last, gene-corrected T cells from CTLA-4-/- mice engrafted and prevented lymphoproliferation in an in vivo murine model of CTLA-4 insufficiency. These results demonstrate the feasibility of a therapeutic approach using T cell gene therapy for CTLA-4 insufficiency

TNF signalling drives expansion of bone marrow CD4+ T cells responsible for HSC exhaustion in experimental visceral leishmaniasis

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These finding provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously

<i>L</i>. <i>donovani</i> infection expands the population of BM T cells expressing IFNγ and TNF.

<p>Comparison of BM T cells in naïve and d28-infected mice (Ld28). (A) Number of T cells in BM. (B) Frequency of total CD44^high and CD44^high subsets within total BM CD4⁺ T cell population. (C) Frequency of IFNγ⁺ subsets in total BM cells. (D) Frequency of TNF⁺ subsets in total BM cells. (E) Frequency of IFNγ⁺ within BM CD4⁺ T cell population following stimulation <i>in vitro</i>. (F) Frequency of IFNγ⁺ within BM CD4⁺ T cell population directly <i>ex vivo</i>. (G) Frequency of TNF⁺ within BM CD4⁺ T cell population following stimulation <i>in vitro</i>. (H) Frequency of TNF⁺ within BM CD4⁺ T cell population directly ex vivo. (I) Mean Fluorescence Intensity (MFI) of IFNγ in IFNγ⁺CD4⁺ T cells. (J) MFI of TNF in TNF⁺CD4⁺ T cells. Data from at least two independent experiments (n = 6–14 per group) presented as scatter plot and mean bar; *p ≤ 0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001; unpaired t test. (K) Representative dot plots for IFNγ expression by stimulated BM CD4⁺ T cells (top) or <i>ex vivo</i> (bottom).</p

Intrinsic IFNγ receptor signaling is required for expansion of BM T cells following infection.

<p>(A) Experimental design for competitive mixed BM chimeras using wild-type (WT) and <i>Ifnγr2</i> knockout (IFNγR2 KO). Analyses were performed 12 weeks after BMT from CD45.2 <i>Ifnr2</i>^-/- mice and CD45.1 WT mice (50:50) to lethally irradiated CD45.1 recipient mice, subsequently infected with <i>L</i>. <i>donovani</i> for 28 days. (B) Frequency of donor cells in BM. (C) Frequency of BM LSK CD150⁺ CD48^- cells (enriched for LT-HSCs) and LSK CD150⁺ CD48⁺ cells within donor cells. (D) Frequency of LT-HSCs in G0 (Ki67^-) within donor cells. (E) Frequency of donor cells in spleen. (F) Frequency of T cells within donor cells in spleen. (G) Number of donor T cells in spleen. (H) Frequency of BM T cells within donor cells. (I) Number of donor T cells in the BM. Data presented as scatter plot and mean bar (n = 4–8); *p ≤ 0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001; One-way Anova followed by Tukey’s multiple comparisons test.</p

Loss of quiescent LT-HSCs following <i>L</i>. <i>donovani</i> infection.

<p>(A and B) Frequency (A) and number (B) of Ki67⁺ within HSPCs populations in BM of naïve and d28-infected B6 mice. (C) Number of LT-HSCs in G0 (Ki67^-) in BM in naïve and d28-infected mice (n = 12 per group; three independent experiments). Data presented as scatter plot and mean bar; unpaired t test; *p ≤ 0.05, **p ≤0.01 ****p ≤0.0001. (D) Representative dot plots for Ki67 expression on LSK CD150⁺CD34^-CD48^- cells.</p

Quiescent LT-HSCs are retained in infected <i>Rag2</i>^-/- mice.

<p>(A) Number of HSPCs in BM of naïve B6.WT (light squares), naïve B6.<i>Rag2</i>^-/- (dark grey triangle), d28 infected B6 (dark grey squares) and d28 <i>Rag2</i>^-/- (light grey circles) mice. (B) Number of BM LT-HSCs in G0 (Ki67^-). (C) Spleen parasite burden in B6.WT and B6 <i>Rag2</i>^-/-, presented as number of parasites per 1000 nuclei. Data from three independent experiments presented as scatter plot and mean bar (n = 9–16 per group); *p ≤ 0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001; unpaired t test.</p

CD4⁺ T cells drive HSC exhaustion in an IFNγ-dependent manner.

<p>(A) Diagram of experimental layout, RAG2 KO mice were adoptively transferred with sorted CD4⁺ T cells from naive mice, and then infected in the following day with <i>L</i>. <i>donovani</i>. At day 28 p.i., we analysed the distribution of hematopoietic progenitors in the BM in naïve RAG mice, infected RAG2 KO mice and infected RAG mice that receive adoptively transferred CD4⁺ T cells: (B-E) Number of HSPCs: LT-HSCs (B), LSK CD150⁺CD34^-CD48⁺ cells (C), LSK CD150⁺CD34⁺ cells (D) and quiescent LT-HSCs LSK in BM (E) (n = 12–17 per group, from three independent experiments). (F) Frequency of HSPCs populations within Lineage negative cells in naïve RAG mice with and without adoptive CD4⁺ T cell transfer; and (F) number of quiescent LT-HSCs (n = 9–5 per group). (H-J) Frequency of progenitor cells within Lineage negative cells in infected RAG mice without or with adoptive transfer of IFNγ sufficient or IFNγ-deficient CD4⁺ T cells (G); Number of quiescent LT-HSCs (n = 4–5) (H). (J) Parasites per 1000 nuclei in the spleen. Data presented as presented as scatter plot and mean bar; *p ≤ 0.05, **p ≤0.01, ***p ≤0.001 and ****p ≤0.0001; one-way Anova and Tukey’s multiple comparisons test.</p

HSPCs from infected mice have defective capacity for self-renewal.

<p>(A) Diagram of experimental layout: naïve lethally irradiated B6.CD45.1xCD45.2 (n = 4) recipients received a 50:50 mix of BM Lin^- cells from d28-infected B6.CD45.2 and naïve B6.CD45.1 mice (A-D); (B) Frequency of donor hematopoietic cells in BM and spleen of recipient mice at 12 weeks post BMT; (C) Frequencies of donor leucocytes in spleen; (D) Frequencies of donor non-committed hematopoietic progenitor cells in BM; (E) Frequencies of lineage-committed progenitors in BM derived from each donor. (F) Diagram of experimental layout: naïve lethally irradiated B6.CD45.1 recipients received a radioprotective dose of 3x10⁵ CD45.1 cells along with 160 HSCs isolated from naïve or d28 infected B6.CD45.2 mice (F-H). (G) Number of donor hematopoietic cells in the BM and in the spleen of recipient mice at 16 weeks post BMT; (H) Number of donor non-committed multipotent progenitors cells in the BM of recipient mice. (I-J) Naïve irradiated B6.CD45.1 recipients received a radio protective dose of 3x10⁵ CD45.1 cells along with 50 HSCs of each origin derived from recipients (n = 3–4 per group) as shown in (F); (I) Number of donor hematopoietic cells in the BM and spleen of recipient mice at 24 weeks post BMT; (J) Number of donor non-committed multipotent progenitors cells in BM. Data presented as scatter plot and mean bar; *p ≤ 0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001 by unpaired t test.</p