Diş pulpası kök hücrelerinin in vitro epitelyal ve fibroblastik hücrelere farklılaşması

Hücresel tedavi ve doku/organ mühendisliği uygulamaları için diş pulpası kaynaklı kök hücreler (DP-KH) önemli bir potansiyele sahiptir. Bu çalışmanın amacı, önceki çalışmalarda nörojenik, adipojenik, miyojenik, odontojenik/osteojenik farklılaşmaları gösterilen iDP-MKH'lerin fibroblastik ve epitelyal hücre dizilerine farklılaşma potansiyelinin belirlenmesidir. Bu araştırmada, lokal anestezi altında çekilen gömük yirmi yaş dişleri (n=5) kullanılmıştır. Açığa çıkarılmış olan pulpa dokusu kollajenaz ile sindirilme işleminden geçirilerek ayrıştırılan hücreler uygun besi ortamında kültüre alınmıştır. Her pasaj sonrası (P1'den P5'e kadar) akış sitometri cihazında ve immunohistokimyasal (İHK) yöntemler ile immunofenotipleme çalışmaları gerçekleştirilmiştir. Bu çalışmalar sonucunda, iDP-MKH'lerin hematopoetik hücre belirteçlerini (CD14, CD34, CD45, CD117, HLA-DR gibi) eksprese etmedikleri, buna karşın MKH belirteçleri CD13, CD44 ve CD90 için yüksek düzeylerde pozitif oldukları tespit edilmiştir. Ayrıca, iDP-MKH'lerin vimentin, fibronektin, ? -düz kas aktin, beta-tubulin, osteokalsin, osteonektin ve embriyonik kök hücre belirteci SSEA-4 eksprese ettikleri gözlenmiştir. Bununla birlikte, iDP-MKH'lerin in vitro koşullarda osteojenik ve adipojenik hücre serilerine farklılaşabildikleri görülmüştür. Osteojenik farklılaşmış hücreler osteokalsin, ostenektin ve BMP4 için kuvvetli pozitif immun reaksiyon göstermiş ve adipositlere farklılaşan iDP-MKH'lerin sitoplazmalarında yağ vakuolleri tespit edilmiştir. Epitelyal farklılaşması EGF, HGF, KGF ve IGF-2 büyüme faktörleri ile uyarılan iDP-MKH'lerin kübik morfolojik farklılaşma ve epitelyal hücre belirteçleri (sitokeratin 18 ve 19) için kuvvetli immun-reaksiyon gösterdikleri buna karşılık nestin ekspresyonlarının azaldığı tespit edilmiştir. Ayrıca, epitel-benzeri hücrelerde vimentin ekspresyonunda artış gözlenmiştir. Fibroblastik farklılaşması bFGF+TGFß ve EGF+ TGFß ile uyarılan iDP-MKH'lerde, fibronektin, fibroblast yüzey proteini, vimentin ve tenaskin-C için kuvvetli pozitif buna karşılık CD105 ve tip II kollajen için negatif immun-reaksiyon tespit edilmiştir. Nestin ekspresyonlarında ise belirgin bir azalma gözlenmiştir. Epitelyal ve fibroblastik farklılaşma gruplarının tümünde, telomeraz aktivitelerinde gerçekleşen azalma ve üremedeki yavaşlık farklılaşan iDP-MKH'lerin somatik hücre karakteristiğine sahip olduğunu göstermiş ve iDP-MKH'lerin in vitro epitelyal ve fibroblastik farklılaşmasını desteklemiştir. Sonuç olarak, bu çalışmada elde edilen veriler iDP-MKH'lerin epitelyal ve fibroblastik farklılaşmalarının in vitro koşullarda uygun uyaranlarla yönlendirilebileceğini ve bu hücrelerin hücre tabanlı klinik tedaviler ve doku mühendisliği uygulamaları için uygun alternatif bir kaynak olabileceğini göstermektedir

HIF-1α hampers dendritic cell function and Th1 generation during chronic visceral leishmaniasis

International audienceInflammation, although responsible for controlling infection, is often associated with the pathogenesis of chronic diseases. Leishmania donovani, the causative agent of visceral leishmaniasis, induces a strong inflammatory response that leads to splenomegaly and ultimately immune suppression. Inflamed tissues are typically characterized by low levels of oxygen, a microenvironment that triggers the hypoxia-inducible transcription factor 1α (HIF-1α). Although HIF-1α plays an integral role in dendritic cell function, its involvement in the generation of protective Th1 responses against Leishmania has not yet been studied. Here we demonstrate that HIF-1α inhibits IL-12 production in dendritic cells, limiting therefore Th1 cell development. Indeed, depletion of HIF-1α in CD11c+ cells resulted in higher and sustained expression of IL-12 and complete abrogation of IL-10. Moreover, CD11c-specific HIF-1α-deficient mice showed higher frequencies of IFN-γ-producing CD4 T cells in the spleen and bone marrow and, consequently, a significantly reduced parasite burden in both organs. Taken together, our results suggest that HIF-1α expression in dendritic cells largely contributes to the establishment of persistent Leishmania infection and may therefore represent a possible therapeutic target

HIF-1α impairs monocyte’s effector function and induces M2 macrophage polarization during chronicLeishmania donovani infection

Leishmaniadonovani impairs the immune responseby triggering a hypoxic micro-environment that facilitates the establishment of a chronic infection. Under this condition, HIF-1α, the master regulator of the response to lowoxygen tension, is stabilized inall cell populations. The effect of hypoxia on the immune response to Leishmania has not yet been investigated. Particularly, the role of HIF-1α in macrophage and monocyte function during chronic visceral leishmaniasis remains unknown. Here, we investigate the role of HIF-1α in inflammatory monocyte and dendritic cell functions, and in the modulation of macrophage polarization during chronic L. donovani infection. To this end, we generated CD11c-specific HIF-1α knock-out mice that were then infected with L. donovani. Our data demonstrate that HIF-1α limits inflammatory monocyte recruitment to the spleen and induces M2 macrophage polarization in response to extracellular lactate. The ablation of HIF-1α in CD11c+ cells resulted in decreased intracellular lactate concentrations, lower expression of M2 macrophage markers, greater expansion and effector capacity of monocytes, and enhanced IFNϒ+ production by CD4+ T cells. Moreover, mice with a targeted depletion of HIF-1α in CD11c+ cells had a significantly lower splenic parasite burden, suggesting that induction of HIF-1α in CD11c+ cells may represent a key factor adopted by Leishmania parasites to establish persistent infections

Infection-adapted emergency hematopoiesis promotes visceral leishmaniasis

<div><p>Cells of the immune system are derived from hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs become activated in response to stress, such as acute infections, which adapt the bone marrow output to the needs of the immune response. However, the impact of infection-adapted HSC activation and differentiation on the persistence of chronic infections is poorly understood. We have examined here the bone marrow outcome of chronic visceral leishmaniasis and show that the parasite <i>Leishmania donovani</i> induces HSC expansion and skews their differentiation towards non-classical myeloid progenitors with a regulatory phenotype. Our results further suggest that emergency hematopoiesis contributes to the pathogenesis of visceral leishmaniasis, as decreased HSC expansion results in a lower parasite burden. Conversely, monocytes derived in the presence of soluble factors from the infected bone marrow environment are more permissive to infection by <i>Leishmania</i>. Our results demonstrate that <i>L</i>. <i>donovani</i> is able to subvert host bone marrow emergency responses to facilitate parasite persistence, and put forward hematopoiesis as a novel therapeutic target in chronic infections.</p></div

Bone marrow HSCs switch their differentiation towards non-classical myeloid progenitors.

<p><b>(A)</b> Representative flow cytometry data and gating strategy of granulocyte-monocyte progenitors (GMPs) in the bone marrow. Steady state myeloid progenitor (MP) cells were first gated on Lin^-Sca1^-c-kit^hi and then subdivided according to the expression of CD41, CD150 and CD16/CD32. GMPs were identified as CD16/CD32⁺ CD41^- CD150^-. Due to the inflammation-induced shift in Sca1 expression, the total Lin^- c-Kit^hi HSPC population was included for analysis during infection. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s002" target="_blank">S2 Fig</a>. <b>(B)</b> Graphs show percentage and absolute numbers of GMPs at various time points. Data are pooled from three independent experiments, with each individual dot representing one mouse. Horizontal lines represent the sample mean. <b>(C)</b> Percentage of GMPs in S/G2/M phases of cell cycle. <b>(D)</b> Percentage of Sca-1⁺ emergency GMPs within all GMPs. <b>(E)</b> Numbers of cells within Sca-1⁺ and Sca-1^- GMP subsets. <b>(F)</b> Intracellular active β-catenin levels (MFI) in GMPs.</p

Diminished myeloid output in <i>Fzd6</i>^-/- mice correlates with a reduced parasite burden during the chronic phase of infection.

<p><b>(A)</b> Analysis of bone marrow myeloid subsets infected <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) mice. Mean percentage for each cell subset is indicated within flow cytometry plots. Graphs show numbers of granulocytes and monocytes on day 14 and 28. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s004" target="_blank">S4</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s006" target="_blank">S6</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s007" target="_blank">S7</a> Figs. <b>(B)</b> Numbers within flow cytometry plots indicate mean percentage of Ly6C^lo/- F4-80⁺ bone marrow macrophages. Histograms show total numbers of macrophages and percent F4-80⁺ within Ly6C^hi monocytes (mean + SEM from seven mice per group). <b>(C)</b> Ly6C and CCR expression (MFI) on Ly6C^hi monocytes at day 28pi. <b>(D)</b> Percentage of CXCR4⁺, Sca-1⁺ and MHC-II⁺ cells within Ly6C^hi monocytes on day 28pi. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s008" target="_blank">S8 Fig</a>. <b>(E)</b> Percentage of Arginase-1 (Arg-1) and IL-10 expressing cells within Ly6C^hi monocytes. <b>(F)</b> NOS2 expression (MFI) on Ly6C^hi monocytes and Ly6C^lo/- F4-80⁺ bone marrow macrophages at day 28pi. <b>(G)</b> Parasite burden determined by the limiting dilution assay in WT and KO bone marrow at day 28. Data shown were pooled from two independent infections with 10 mice per genotype. <b>(H-I)</b> Pearson's correlation coefficient was used to assess correlation between bone marrow parasite burden and bone marrow HSCs <b>(H)</b> and Ly6C^hi monocytes <b>(I)</b> during the course of the infection. Data for correlation were pooled from C57BL/6, <i>Fzd6</i>^+/+ and <i>Fzd6</i>^-/- mice at day 14, 21 or 28. All bar graphs represent mean + SEM with 6 mice per group for day 28 and 3 mice per group for day 14 unless otherwise noted. Similar results were obtained from three independent experiments for day 28. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p

<i>Fzd6</i>^-/- T lymphocytes are functionally indistinguishable from their <i>Fzd6</i>^+/+ counterparts.

<p><b>(A-B)</b> Numbers of CD4⁺ and CD8⁺ T cells in BM <b>(A)</b> and spleen <b>(B)</b> of naïve and infected mice on day 28. <b>(C-D)</b> Cytokine production by bone marrow and spleen CD4⁺ T cells isolated from infected mice and stimulated <i>ex vivo</i> with parasite-pulsed bone marrow dendritic cells. Representative flow cytometry data are shown in <b>(C)</b>. Graph in <b>(D)</b> shows compiled results from a representative experiment. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s010" target="_blank">S10 Fig</a>. All bar graphs represent mean + SEM with 7 mice per group for day 28. <b>(E)</b> Macrophages derived from naïve <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) bone marrow were either left untreated, stimulated with IFN-γ alone or first primed with IFN-γ and then infected with PKH26-labeled <i>L</i>. <i>donovani</i> amastigotes. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s010" target="_blank">S10 Fig</a>. Imaging flow cytometry analysis of macrophages infected with fluorescent <i>Leishmania</i> parasites, showing multiple parasites within both <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) macrophages. <b>(F)</b> Histograms represent percentage of infected macrophages and <b>(G)</b> parasite numbers in infected macrophages 72h post-infection. Low = 1–3, medium = 4–10, high = >10 parasites / cell. Bar graphs represent mean + SEM from two independent experiments.</p

Infected bone marrow microenvironment directly promotes HSPC expansion and the generation of permissive monocytes.

<p>Freshly isolated lineage-depleted <i>Fzd6</i>^+/+ (WT) BM cells were cultured in complete medium supplemented with 30% BM supernatant as indicated. <b>(A)</b> Representative flow cytometry data show the gating strategy for CD11b⁺, LSK and GMP populations. Graphs show numbers of cell recovered per 5x10⁵ cells seeded for each subset. Lin^- <i>Fzd6</i>^+/+ (WT) BM cells cultured with BM supernatant obtained from infected <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) mice. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s011" target="_blank">S11 Fig</a>. Data were pooled from three independent experiments. <b>(B)</b> Percentage of Ly6C⁺ monocytes in differentiation cultures following infection on day 4, and <b>(C)</b> the proportion of infected Ly6C⁺ monocytes after 24h and 72h. <b>(D-E)</b> Imaging flow cytometry analysis of monocytes infected with fluorescent Leishmania parasites, showing multiple parasites at <b>(D)</b> 24h and <b>(E)</b> 72h post-infection. Histograms represents parasite uptake in infected macrophages. Low = 1–3, medium = 4–10, high = >10 parasites respectively. Bar graphs represent mean + SEM from three independent experiments. *<i>P</i><0.05; **<i>P</i><0.01.</p

Decreased accumulation of myeloid cells is accompanied with reduced parasite burden in <i>Fzd6</i>^-/- spleen.

<p><b>(A)</b> Analysis of myeloid subsets in the spleen of infected <i>Fzd6</i>^-/- (KO) and <i>Fzd6</i>^+/+ (WT) mice. Mean percentage for each cell subset is indicated within flow cytometry plots. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s007" target="_blank">S7</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s008" target="_blank">S8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006422#ppat.1006422.s009" target="_blank">S9</a> Figs. <b>(B)</b> Graphs show numbers of granulocytes and monocytes on day 14 and 28. <b>(C)</b> F4-80⁺ cells within Ly6C^hi monocytes and numbers of Ly6C^lo/- F4-80⁺ macrophages in the spleen. <b>(D)</b> Parasite burden expressed as Leishmania Donovani Units (LDU) in spleen on days 14 and 28pi. <b>(E)</b> Ratio of splenic to bone marrow granulocytes and Ly6C^hi monocytes in infected KO and WT mice on day 28 (mean + SEM from six mice per group). <b>(F)</b> Percentage of Sca-1⁺ and MHC-II⁺ cells within Ly6C^hi monocytes on day 28pi. <b>(G)</b> Ly6C and CCR2 expression (MFI) on Ly6C^hi monocytes at day 28pi. <b>(H)</b> Percentage of IL-10 expressing cells and NOS2 expression (MFI) on Ly6Chi monocytes at day 28pi. All bar graphs represent mean + SEM with 18 mice per group for day 28 pooled from three independent experiments and 3 mice per group for day 14 unless otherwise noted. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p