Pro-inflammatory cytokine blockade attenuates myeloid expansion in a murine model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease characterized by chronic inflammation and progressive destruction of joint tissue. It is also characterized by aberrant blood phenotypes including anemia and suppressed lymphopoiesis that contribute to morbidity in RA patients. However, the impact of RA on hematopoietic stem cells (HSC) has not been fully elucidated. Using a collagen-induced mouse model of human RA, we identified systemic inflammation and myeloid overproduction associated with activation of a myeloid differentiation gene program in HSC. Surprisingly, despite ongoing inflammation, HSC from arthritic mice remain in a quiescent state associated with activation of a proliferation arrest gene program. Strikingly, we found that inflammatory cytokine blockade using the interleukin-1 receptor antagonist anakinra led to an attenuation of inflammatory arthritis and myeloid expansion in the bone marrow of arthritic mice. In addition, anakinra reduced expression of inflammation-driven myeloid lineage and proliferation arrest gene programs in HSC of arthritic mice. Altogether, our findings show that inflammatory cytokine blockade can contribute to normalization of hematopoiesis in the context of chronic autoimmune arthritis.

PU.1 is required to restrain myelopoiesis during chronic inflammatory stress.

Chronic inflammation is a common feature of aging and numerous diseases such as diabetes, obesity, and autoimmune syndromes and has been linked to the development of hematological malignancy. Blood-forming hematopoietic stem cells (HSC) can contribute to these diseases via the production of tissue-damaging myeloid cells and/or the acquisition of mutations in epigenetic and transcriptional regulators that initiate evolution toward leukemogenesis. We previously showed that the myeloid "master regulator" transcription factor PU.1 is robustly induced in HSC by pro-inflammatory cytokines such as interleukin (IL)-1ß and limits their proliferative activity. Here, we used a PU.1-deficient mouse model to investigate the broader role of PU.1 in regulating hematopoietic activity in response to chronic inflammatory challenges. We found that PU.1 is critical in restraining inflammatory myelopoiesis via suppression of cell cycle and self-renewal gene programs in myeloid-biased multipotent progenitor (MPP) cells. Our data show that while PU.1 functions as a key driver of myeloid differentiation, it plays an equally critical role in tailoring hematopoietic responses to inflammatory stimuli while limiting expansion and self-renewal gene expression in MPPs. These data identify PU.1 as a key regulator of "emergency" myelopoiesis relevant to inflammatory disease and leukemogenesis.

Platelets from blood diversion pouches (DPs) are a suitable alternative for functional, bioenergetic, and metabolomic analyses.

BACKGROUND: The collection of the first blood flow into a diversion pouch (DP) has become widely adopted in blood donation systems to reduce whole-blood unit contamination from skin bacteria. The strict control of pre-analytical variables, such as blood collection and proper anticoagulant selection, is critical to diminish experimental variability when studying different aspects of platelet biology. We hypothesize that the functional, mitochondrial, and metabolomic profiles of platelets isolated from the DP are not different from the ones isolated from standard venipuncture (VP), thus representing a suitable collection method of platelets for experimental purposes. MATERIALS AND METHODS: Whole blood from the blood DP or VP was collected. Platelets were subsequently isolated and washed following standard protocols. Platelet function was assessed by flow cytometry, light transmission aggregometry, clot retraction, and under flow conditions using the total thrombus formation analyzer (T-TAS). Mitochondrial function and the platelet metabolome profiles were determined by the Seahorse extracellular flux analyzer (Agilent, Santa Clara, CA, USA) and ultra-high-pressure liquid chromatography-mass spectrometry metabolomics, respectively. RESULTS: Platelets isolated from VP and the DP have similar functional, mitochondrial, and metabolic profiles with no significant differences between both groups at baseline and upon activation by any of the assays mentioned above. DISCUSSION: The findings of our study support the use of platelets from the DP for performing functional and metabolic studies on platelets from a wide range of blood donors. The DP may serve as an alternative blood collection method to standard VP, allowing the study of diverse aspects of platelet biology, such as age, sex, race, and ethnicity, in many eligible individuals for blood donation.

Expression of acetylated histones H3 and H4 and histone deacetylase enzymes HDAC1, HDAC2 and HDAC6 in simple mammary carcinomas of female dogs.

Histone deacetylation is an important mechanism involved in human breast cancer tumorigenesis and recent veterinary oncology studies also demonstrate a similar relationship in some canine neoplasms. The use of HDAC inhibitors in vitro and in vivo has demonstrated antitumor action on several strains of human and animal cancers. The present study aims to correlate the expression of H3K9Ac, H4K12Ac, HDAC1, HDAC2 and HDAC6 in simple mammary carcinomas in dogs with clinicopathological parameters and overall survival time. To this end, 61 samples of simple breast carcinomas were analyzed by the immunohistochemistry technique with subsequent validation of the antibodies by the Western Blot technique. The expressions obtained via a semi-quantitative way were categorized by assigning scores and classified into high or low expressions according to the given score, except for HDAC6, when the marking percentage was considered and subdivided into high and low expressions using the median value. For statistical analysis, the chi-square test or Fisher exact test were used as univariate analysis and correspondence analysis as a multivariate test, in addition to the Kaplan-Meier survival analysis. In the studied samples, the highest frequencies were determined for the high expression proteins H4K12Ac (88.5%), HDAC2 (65.6%) and HDAC6 (56.7%) and the low expression proteins H3K9Ac (73.8%) and HDAC1 (54.1%). An association between the low expression of HDAC1 and the presence of lymph node metastasis (p = 0.035) was indicated by univariate analysis while the high expression of HDAC1 was associated with favorable prognostic factors, such as the absence of lymph node metastasis and low mitotic index by multivariate analysis. Also, by multivariate analysis, the low expression of HDAC6 was correlated with the low expression of Ki67, smaller tumors, and better prognosis factors as well. Protein expression was not correlated with patients' overall survival time (p > 0.05). The high expressions of HDAC2 and HDAC6 in mammary carcinomas in female dogs may be useful information for research involving therapeutic targets with iHDACs since their inhibition favors hyperacetylation and transcription of tumor suppressor genes.

PU.1 Expression Defines Distinct Functional Activities in the Phenotypic HSC Compartment of a Murine Inflammatory Stress Model.

The transcription factor PU.1 is a critical regulator of lineage fate in blood-forming hematopoietic stem cells (HSC). In response to pro-inflammatory signals, such as the cytokine IL-1ß, PU.1 expression is increased in HSC and is associated with myeloid lineage expansion. To address potential functional heterogeneities arising in the phenotypic HSC compartment due to changes in PU.1 expression, here, we fractionated phenotypic HSC in mice using the SLAM surface marker code in conjunction with PU.1 expression levels, using the PU.1-EYFP reporter mouse strain. While PU.1lo SLAM cells contain extensive long-term repopulating activity and a molecular signature corresponding to HSC activity at steady state, following IL-1ß treatment, HSCLT induce PU.1 expression and are replaced in the PU.1lo SLAM fraction by CD41+ HSC-like megakaryocytic progenitors (SL-MkP) with limited long-term engraftment capacity. On the other hand, the PU.1hi SLAM fraction exhibits extensive myeloid lineage priming and clonogenic activity and expands rapidly in response to IL-1ß. Furthermore, we show that EPCR expression, but not CD150 expression, can distinguish HSCLT and SL-MkP under inflammatory conditions. Altogether, our data provide insights into the dynamic regulation of PU.1 and identify how PU.1 levels are linked to HSC fate in steady state and inflammatory stress conditions.

PU.1 enforces quiescence and limits hematopoietic stem cell expansion during inflammatory stress.

Hematopoietic stem cells (HSCs) are capable of entering the cell cycle to replenish the blood system in response to inflammatory cues; however, excessive proliferation in response to chronic inflammation can lead to either HSC attrition or expansion. The mechanism(s) that limit HSC proliferation and expansion triggered by inflammatory signals are poorly defined. Here, we show that long-term HSCs (HSCLT) rapidly repress protein synthesis and cell cycle genes following treatment with the proinflammatory cytokine interleukin (IL)-1. This gene program is associated with activation of the transcription factor PU.1 and direct PU.1 binding at repressed target genes. Notably, PU.1 is required to repress cell cycle and protein synthesis genes, and IL-1 exposure triggers aberrant protein synthesis and cell cycle activity in PU.1-deficient HSCs. These features are associated with expansion of phenotypic PU.1-deficient HSCs. Thus, we identify a PU.1-dependent mechanism triggered by innate immune stimulation that limits HSC proliferation and pool size. These findings provide insight into how HSCs maintain homeostasis during inflammatory stress.

CD34 and EPCR coordinately enrich functional murine hematopoietic stem cells under normal and inflammatory conditions.

Hematopoiesis is dynamically regulated to maintain blood system function under nonhomeostatic conditions such as inflammation and injury. However, common surface marker and hematopoietic stem cell (HSC) reporter systems used for prospective enrichment of HSCs have been less rigorously tested in these contexts. Here, we use two surface markers, EPCR/CD201 and CD34, to re-analyze dynamic changes in the HSC-enriched phenotypic SLAM compartment in a mouse model of chronic interleukin (IL)-1 exposure. EPCR and CD34 coordinately identify four functionally and molecularly distinct compartments within the SLAM fraction, including an EPCR+/CD34- fraction whose long-term serial repopulating activity is only modestly impacted by chronic IL-1 exposure, relative to unfractionated SLAM cells. Notably, the other three fractions expand in frequency following IL-1 treatment and represent actively proliferating, lineage-primed cell states with limited long-term repopulating potential. Importantly, we find that the Fgd5-ZSGreen HSC reporter mouse enriches for molecularly and functionally intact HSCs regardless of IL-1 exposure. Together, our findings provide further evidence of dynamic heterogeneity within a commonly used HSC-enriched phenotypic compartment under stress conditions. Importantly, they also indicate that stringency of prospective isolation approaches can enhance interpretation of findings related to HSC function when studying models of hematopoietic stress.

Calidad de vida relacionada con la salud en adultos con normopeso, sobrepeso y obesidad / Health-related quality of life in normal weight, overweight and obese adults

Resumen Actualmente el sobrepeso y la obesidad presentan una alta prevalencia a nivel mundial, y se encuentran relacionados con enfermedades crónico-degenerativas, mortalidad prematura y disminución en la calidad de vida. El propósito de esta investigación fue comparar la calidad de vida relacionada con la salud (CVRS) en función del estado de nutrición (EN) y sexo. Participaron 202 adultos (84 hombres y 118 mujeres), con una edad entre 18 y 81 años (M =39.74 DE = 13.77), quienes fueron distribuidos por su EN en: normopeso, sobrepeso y obesidad. Los participantes completaron el cuestionario 36-Item Short Form Health Survey (SF-36). En la muestra predominó la dimensión Función física (FF), considerándola muy buena. Las dimensiones Rol emocional (RE), Rol físico (RF), Vitalidad (VT) y Salud mental (SM) se ubicaron en el nivel de bueno, y solamente la Salud general (SG) se consideró regular. En el caso de la comparación entre los grupos en función del EN no se encontraron diferencias significativas; sin embargo, en la comparación por sexo, los hombres obtuvieron mayores puntuaciones en: FF, SM, VT y RE. Se concluye que no hay diferencia estadísticamente significativa de la CVRS entre los grupos por EN, no obstante, al realizar la comparación por sexo se encuentran diferencias significativas a favor de los hombres.

Abstract Overweight and obesity currently have a high prevalence worldwide, are related to chronic degenerative diseases, premature mortality and the decline in quality of life. The purpose of the present investigation was to compare the quality of life related to health (HRQoL) between groups by their nutritional status (NS) and by sex. There was a sample of 202 participants (84 men and 118 women) with an age between 18 and 81 years (M =39.74 SD = 13.77), distributed by their NS in: normal weight, overweight and obesity, they were administered the questionnaire 36-Item Short Form Health Survey (SF-36). In the general sample, the Physical Function (PF) dimension was predominant, being considered as very good, the scores obtained in the Emotional Role (ER), Physical Role (PR), Vitality (VT) and Mental Health (MH) dimensions were in a range estimated as good and only the General Health (GH) dimension was considered regular. In the case of the comparison between the groups by state of nutrition, no statistically significant differences were found between them, however, in the comparison made by sex, statistically significant differences were found in the factors of PF, MH, VT and ER in favor of the group of men. In conclusion, there is not statistically significant difference of the HRQoL between the groups by NS, nevertheless, when performing the comparison by sex, significant differences are found in favor of the men.

Ehrlichia canis (Jaboticabal strain) induces the expression of TNF-α in leukocytes and splenocytes of experimentally infected dogs.

Canine ehrlichiosis is caused by the bacterium Ehrlichia canis and is characterized by a systemic febrile disease of unknown pathogenesis. This study evaluated the expression of cytokines TNF-α, IL-10, IFN-γ, in splenic cells and blood leukocytes during the acute phase of ehrlichiosis and after treatment with doxycycline hyclate in dogs experimentally infected with the E. canis Jaboticabal strain. The study results showed a significant expression of TNF-α 18 days post-inoculation, reducing by approximately 70% after treatment. There was a unique peak of expression of IL-10 and IFN-γ 18 and 30 days post-inoculation, respectively. This study suggests that TNF-α plays a role in the pathogenesis of the acute phase of canine ehrlichiosis and that treatment with doxycycline hyclate reduces the systemic effects of this cytokine, possibly by reducing or eliminating parasitemia.

Ehrlichia canis (Jaboticabal strain) induces the expression of TNF-α in leukocytes and splenocytes of experimentally infected dogs / Amostra Ehrlichia canis (Jaboticabal) induz a expressão de TNF-α em leucócitos e esplenócitos de cães experimentalmente infectados

Canine ehrlichiosis is caused by the bacterium Ehrlichia canis and is characterized by a systemic febrile disease of unknown pathogenesis. This study evaluated the expression of cytokines TNF-α, IL-10, IFN-γ, in splenic cells and blood leukocytes during the acute phase of ehrlichiosis and after treatment with doxycycline hyclate in dogs experimentally infected with the E. canis Jaboticabal strain. The study results showed a significant expression of TNF-α 18 days post-inoculation, reducing by approximately 70 percent after treatment. There was a unique peak of expression of IL-10 and IFN-γ 18 and 30 days post-inoculation, respectively. This study suggests that TNF-α plays a role in the pathogenesis of the acute phase of canine ehrlichiosis and that treatment with doxycycline hyclate reduces the systemic effects of this cytokine, possibly by reducing or eliminating parasitemia.

A erliquiose canina é causada pela bactéria Ehrlichia canis, que desencadeia no hospedeiro uma doença febril e sistêmica, de patogênese pouco conhecida. O presente estudo avaliou a expressão das citocinas TNF-α, IL-10, IFN-γ, em células esplênicas e em leucócitos sanguíneos, durante a fase aguda da erliquiose e após o tratamento com hiclato de doxiciclina, em cães experimentalmente infectados com a amostra E. canis Jaboticabal. Os resultados mostraram expressão significativa de TNF-α 18 dias após a inoculação, reduzindo aproximadante 70 por cento após o tratamento. Houve um único pico de expressão de IL-10 e de IFN-γ entre 18 e 30 dias após a inoculação, respectivamente. Este estudo sugere que o TNF-α participa da patogenia da fase aguda da erliquiose canina, e que o tratamento com hiclato de doxiciclina reduz os efeitos sistêmicos dessa citocina, possivelmente por reduzir ou eliminar a parasitemia.