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Supplementary Data from In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target

Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients’ erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV.Significance:Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV.This article is highlighted in the In This Issue feature, p. 265

Supplementary Table from In Vivo Monitoring of Polycythemia Vera Development Reveals Carbonic Anhydrase 1 as a Potent Therapeutic Target

Research question What is the impact of radiation exposure on oocyte quality and female fertility? Design Prepubertal mice underwent whole-body irradiation with a single dose (0.02, 0.1, 0.5, 2, 8 Gy) of gamma- or X-rays. Oocytes were quantified in irradiated (n = 36) and sham-treated (n = 8) mice. After a single exposure to 2 Gy, formation of DNA double-strand breaks (n = 10), activation of checkpoint kinase (Chk2) (n = 10) and dynamics of follicular growth (n = 18) were analysed. Fertility assessment was performed in adult irradiated mice and controls from the number of pups per mouse (n = 28) and the fetal abortion rate (n = 24). Ploidy of mature oocytes (n = 20) was analysed after CREST immunostaining, and uterine sections were examined. Results Radiation exposure induced a massive loss of primordial follicles with LD50 below 50 mGy for both gamma and X-rays. Growing follicles survived doses up to 8 Gy. This difference in radiosensitivity was not due to a different amount of radio-induced DNA damage, and Chk2 was activated in all oocytes. Exposure to a 2 Gy dose abolished the long-term fertility of females due to depletion of the ovarian reserve. Detailed analysis indicates that surviving oocytes were able to complete folliculogenesis and could be fertilized. This transient fertility allowed irradiated females to produce a single litter albeit with a high rate of fetal abortion (23%, P = 0.0096), related to altered ploidy in the surviving oocytes (25.5%, P = 0.0035). Conclusions The effects of radiation on surviving oocyte quality question natural conception as a first-line approach in cancer survivors. Together, the data emphasize the need for fertility preservation before radiation exposure and call for reassessment of the use of cryopreserved oocytes.

Mexico, one of the countries with greatest natural wealth, makes a very attractive commercial partner for the rest of the countries, either for promotion or production processes. In this context, according to the interests of this article, rail transport is mainly used to move goods for import or export. Year by year, in the metropolitan area of Guadalajara, there is an increase in the construction sector that now “represents 11% of the gross domestic product in Jalisco” (Mexican Chamber of the Construction Industry, 2021) and for this, the distribution of various products and raw materials is needed, including heavy machinery and raw materials such as stone (marble), which is difficult to move because of its volume and requires rail transport to be distributed. This research aims to identify through documentary analysis the financial risk factors that affect the distribution of stone (marble) and heavy machinery via rail transport, in companies located in the metropolitan area of Guadalajara, Jalisco. México, uno de los países con mayor riqueza natural, es un potencial socio comercial muy atractivo para el resto de los países, ya sea para procesos de promoción o de producción. En este contexto, de acuerdo con los intereses de este artículo ,para realizar los traslados de mercancías para la importación o para la exportación, se utiliza principalmente el transporte ferroviario. Año a año, en la zona metropolitana de Guadalajara, existe un incremento en el sector de la construcción que ahora “representa el 11% del producto interno bruto en Jalisco” (Camara Mexicana de la Industria de la Construcción, 2021) y para ello se necesita la distribución de varios productos y materia prima, entre ellos maquinaria pesada y materia prima como piedra (mármol), que es difícil de trasladar por su volumen y necesita del transporte férreo para distribuirse. La presente investigación pretende mediante el análisis documental identificar los factores de riesgo financiero que afectan la distribución de piedra (mármol) y la maquinaria pesada vía transporte ferroviario, en empresas ubicadas en la zona metropolitana de Guadalajara, Jalisco.

Current murine models of myeloproliferative neoplasms (MPNs) cannot examine how MPNs progress from a single bone marrow source to the entire hematopoietic system. Thus, using transplantation of knock-in JAK2V617F hematopoietic cells into a single irradiated leg, we show development of polycythemia vera (PV) from a single anatomic site in immunocompetent mice. Barcode experiments reveal that grafted JAK2V617F stem/progenitor cells migrate from the irradiated leg to nonirradiated organs such as the contralateral leg and spleen, which is strictly required for development of PV. Mutant cells colonizing the nonirradiated leg efficiently induce PV in nonconditioned recipient mice and contain JAK2V617F hematopoietic stem/progenitor cells that express high levels of carbonic anhydrase 1 (CA1), a peculiar feature also found in CD34+ cells from patients with PV. Finally, genetic and pharmacologic inhibition of CA1 efficiently suppresses PV development and progression in mice and decreases PV patients’ erythroid progenitors, strengthening CA1 as a potent therapeutic target for PV. Significance: Follow-up of hematopoietic malignancies from their initiating anatomic site is crucial for understanding their development and discovering new therapeutic avenues. We developed such an approach, used it to characterize PV progression, and identified CA1 as a promising therapeutic target of PV. This article is highlighted in the In This Issue feature, p. 265

The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4+ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4+ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure., Author Summary Human Immunodeficiency Virus infection is predominantly transmitted by mucosal exposure, after sexual intercourse. Although substantial progresses have been recently achieved in our understanding of the mechanisms of HIV mucosal transmission, many questions remain. Semen is one of the major sources for HIV which contains both cell-free viral particles and viral infected cells. However, today, the role of cell-associated virus has been largely understudied. We provide here a detailed characterization of the semen leukocyte populations in the highly relevant experiment model of SIV infection of macaques. We demonstrate that the major target cells for the virus, CD4+ T cells and macrophages, are present in macaques semen at all stages of infection. Both cell types are productively infected in vivo and are endowed with adhesion and migration markers that may facilitate virus transmission during sexual exposure. The acute phase of infection is associated with a strong seminal inflammation that may increase semen leukocytes infectivity. This work supports for a role of cell-associated virus in HIV transmission which needs to be considered for the design of prevention strategies.

Background Spontaneous long-term HIV/SIV control in HIV-controller patients and SIV-controller macaques (SIC) is usually associated to protective MHC-class-I alleles and efficient CD8 T-cell responses. However, many HIV-controllers efficiently control HIV-infection despite of non-protective MHC background and weak CD8 T-cell responses, raising the question of real contribution of this response in maintaining viral control. We addressed this question by depleting in vivo CD8+ cells in SIC bearing or not protective MHC and weak CD8 T-cell responses.

Introduction Interferon-α response is one the first innate immune response to HIV/SIV infections during primary infection but is associated with immune suppression, chronic immune activation and pathogenesis. Cell types involved in Interferon alpha production are poorly defined as pDC activation levels are, and reasons for which IFN-α are only transiently detected during acute primary infection, and re-appear in the late clinical AIDS phase are not known. Methods Macaques were infected by SIVmac251 to further analyse the dynamics of pDC and IFN-α during primary infection. We performed a multi-parameter followup: quality and kinetic of IFN-alpha responses (mRNA levels by RT-QPCR, antiviral activity by MDBK/VSV assay, ELISA), define cell types involved in IFN-α production (9 colors flow cytometry), monitor plasmacytoid DCs (pDC) activation/IFN-α production, monitor different pDC precursor stages by flow cytometry, monitor virus loads. Results Plasma viral loads peaked on day 10–11 p.i. to progressively reach a plateau on month 2–3. IFN-α plasma concentrations peaked on day 7–9 p.i. to become rapidly undetectable after day 14–17 p.i.. pDCs were the only cell type with detectable of IFN-α levels in peripheral lymph nodes (PLN) leukocytes. IFN-α mRNA levels correlated both to viral RNA loads and % of IFN-α positive pDCs, which reached transiently 1.82% ±1.52 positive cells on day 9 p.i.. In gut mucosa, IFN-α mRNA levels were also increased in some macaques in association with a raise of BDCA-2 mRNA levels. In PLN a much higher % of pDC transiently show increased expression of activation and apoptosis markers as CCR7, HLA-DR, CD40, CD86, CD95 (on day 9 p.i, 41 ± 13% of pDCs were HLA-DR high CCR7 high and 95 ± 2.93 % were CD95 positive). In parallel we observed an increase of pDC with precursor phenotype (CD123 low HLA-DR low , 22% ± 14 on day 9 p.i.) which express Ki67+ in PLN. This subtype is reminiscent of cells normally present in the bone marrow at steady state which are not able to produce IFN-α in response to SIV in vitro . The proportion of these Ki67+ precursor pDC population circulating in blood correlated with decreased production of IFN-α by pDCs in response to SIV in vitro . Setpoint was associated with a return to baseline of IFN-α mRNA levels in lymphnodes and gut mucosa, and to lower although sustained activation of pDC in lymph nodes, with no detectable IFN-α positive pDCs. Conclusion These data: 1/support that pDC are the main contributors, if not the only, to IFN-α production in response to SIVin this phase. 2/Strongly suggest that the replacement of strongly activated pDC prone to apoptosis by non-functional bone marrow derived early pDC precursors in PLN contribute to rapid decrease of IFN-α production in primary infection, and may contribute to transient decrease of pDC responsiveness to in vitro stimulation. 3/Sustained activation of the pDC pool at set point in the absence of detectable IFN-α expression suggest different threshold of virus loads or imflammation are responsible for differential pDC functions in the chronic compared to primary phase.

A number of men receiving prolonged suppressive highly active antiretroviral therapy (HAART) still shed human immunodeficiency virus (HIV) in semen. To investigate whether this seminal shedding may be due to poor drug penetration and/or viral production by long-lived cells within male genital tissues, we analyzed semen and reproductive tissues from macaques chronically infected with simian immunodeficiency virus mac251 (SIVmac251) who were treated for 4 months with HAART, which was intensified over the last 7 weeks with an integrase inhibitor. We showed that a subset of treated animals continued shedding SIV in semen despite efficient HAART. This shedding was not associated with low antiretroviral drug concentrations in semen or in testis, epididymis, seminal vesicles, and prostate. HAART had no significant impact on SIV RNA in the urethra, whereas it drastically reduced SIV RNA levels in the prostate and vas deferens and to a lesser extent in the epididymis and seminal vesicle. The only detectable SIV RNA-positive cells within the male genital tract after HAART were urethral macrophages. SIV DNA levels in genital tissues were not decreased by HAART, suggesting the presence throughout the male genital tract of nonproductively infected cells. In conclusion, our results demonstrate that 4 months of HAART induced variable and limited control of viral infection in the male reproductive organs, particularly in the urethra, and suggest that infected long-lived cells in the male genital tract may be involved in persistent seminal shedding during HAART. These results pave the way for further investigations of male genital organ infection in long-term-treated infected individuals.A substantial subset of men receiving prolonged HAART suppressing viral loads in the blood still harbor HIV in semen, and cases of sexual transmission have been reported. To understand the origin of this persistence, we analyzed the semen and male reproductive tissues from SIV-infected macaques treated with HAART. We demonstrated that persistent seminal shedding was not linked to poor drug penetration in semen or semen-producing prostate, seminal vesicle, epididymis, and testis. We revealed that HAART decreased SIV RNA to various extents in all male genital organs, with the exception of the urethra, in which SIV RNA(+) macrophages were observed despite HAART. Importantly, HAART did not impact SIV DNA levels in the male genital organs. These results suggest that infection of male genital organs, and particularly the urethra, could be involved in the release of virus in semen during HAART.

Chronic immune activation (IA) is considered as the driving force of CD4+ T cell depletion and AIDS. Fundamental clues in the mechanisms that regulate IA could lie in natural hosts of SIV, such as African green monkeys (AGMs). Here we investigated the role of innate immune cells and IFN-α in the control of IA in AGMs. AGMs displayed significant NK cell activation upon SIVagm infection, which was correlated with the levels of IFN-α. Moreover, we detected cytotoxic NK cells in lymph nodes during the early acute phase of SIVagm infection. Both plasmacytoid and myeloid dendritic cell (pDC and mDC) homing receptors were increased, but the maturation of mDCs, in particular of CD16+ mDCs, was more important than that of pDCs. Monitoring of 15 cytokines showed that those, which are known to be increased early in HIV-1/SIVmac pathogenic infections, such as IL-15, IFN-α, MCP-1 and CXCL10/IP-10, were significantly increased in AGMs as well. In contrast, cytokines generally induced in the later stage of acute pathogenic infection, such as IL-6, IL-18 and TNF-α, were less or not increased, suggesting an early control of IA. We then treated AGMs daily with high doses of IFN-α from day 9 to 24 post-infection. No impact was observed on the activation or maturation profiles of mDCs, pDCs and NK cells. There was also no major difference in T cell activation or interferon-stimulated gene (ISG) expression profiles and no sign of disease progression. Thus, even after administration of high levels of IFN-α during acute infection, AGMs were still able to control IA, showing that IA control is independent of IFN-α levels. This suggests that the sustained ISG expression and IA in HIV/SIVmac infections involves non-IFN-α products., Author Summary Chronic inflammation is considered as directly involved in AIDS pathogenesis. The role of IFN-α as a driving force of chronic inflammation is under debate. Natural hosts of SIV, such as African green monkeys (AGMs), avoid chronic inflammation. We show for the first time that NK cells are strongly activated during acute SIVagm infection. This further demonstrates that AGMs mount a strong early innate immune response. Myeloid and plasmacytoid dendritic cells (mDCs and pDCs) homed to lymph nodes; however mDCs showed a stronger maturation profile than pDCs. Monitoring of cytokine profiles in plasma suggests that the control of inflammation in AGMs is starting earlier than previously considered, weeks before the end of the acute infection. We tested whether the capacity to control inflammation depends on the levels of IFN-α produced. When treated with high doses of IFN-α during acute SIVagm infection, AGMs did not show increase of immune activation or signs of disease progression. Our study provides evidence that the control of inflammation in SIVagm infection is not the consequence of weaker IFN-α levels. These data indicate that the sustained interferon-stimulated gene induction and chronic inflammation in HIV/SIVmac infections is driven by factors other than IFN-α.