Periparturient blood T-lymphocyte PD-1 and CTLA-4 expression as potential predictors of new intramammary infections in dairy cows during early lactation (short communication).

BACKGROUND: The periparturient period in dairy cows is marked by immunosuppression which increases the likelihood of infectious disorders, particularly also mastitis. An in-depth understanding of peripartum leukocyte biology is vital for the implementation of highly successful post-partum disease prevention measures. Immune checkpoint molecules, such as programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4), are critical inhibitory receptors expressed on immune cells, particularly T cells, that drive immunosuppressive signaling pathways. However, the potential role of immune checkpoint molecules expression in T-cells on udder health has never been explored. Thus, the association between the occurrence of new postpartum intramammary infections (IMIs) and the expression of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) on blood T-cells during the peripartum period was investigated. RESULTS: In this study, the incidence of IMIs by any pathogen in early lactation was not associated with a higher expression of PD-1 and CTLA-4 in the periparturient period. However, the incidence of IMIs by major pathogens throughout the first month of lactation was significantly associated with higher expression of PD-1 at 14 days before calving (P = 0.03) and CTLA-4 at parturition (P = 0.03) by blood T-cells. Also, the expression of CTLA-4 at D0 (P = 0.012) by T-cells was associated with the occurrence of persistent IMIs during the first month of lactation. CONCLUSIONS: To our knowledge, this is the first report to investigate the expression of PD-1 and CTLA-4 by blood T-lymphocytes during the periparturient period in dairy cows and to explore their relationship with the incidence of new IMIs in the postpartum period. Thus, a comprehensive understanding of leukocyte biology during peripartum would appear to be a prerequisite for the identification of resilient dairy cows or targets innovative (immunological) non-antibiotic approaches in the transition period.

ATP6V0d2 controls Leishmania parasitophorous vacuole biogenesis via cholesterol homeostasis.

V-ATPases are part of the membrane components of pathogen-containing vacuoles, although their function in intracellular infection remains elusive. In addition to organelle acidification, V-ATPases are alternatively implicated in membrane fusion and anti-inflammatory functions controlled by ATP6V0d2, the d subunit variant of the V-ATPase complex. Therefore, we evaluated the role of ATP6V0d2 in the biogenesis of pathogen-containing vacuoles using ATP6V0d2 knock-down macrophages infected with the protozoan parasite Leishmania amazonensis. These parasites survive within IFNγ/LPS-activated inflammatory macrophages, multiplying in large/fusogenic parasitophorous vacuoles (PVs) and inducing ATP6V0d2 upregulation. ATP6V0d2 knock-down decreased macrophage cholesterol levels and inhibited PV enlargement without interfering with parasite multiplication. However, parasites required ATP6V0d2 to resist the influx of oxidized low-density lipoprotein (ox-LDL)-derived cholesterol, which restored PV enlargement in ATP6V0d2 knock-down macrophages by replenishing macrophage cholesterol pools. Thus, we reveal parasite-mediated subversion of host V-ATPase function toward cholesterol retention, which is required for establishing an inflammation-resistant intracellular parasite niche.

Dual Host-Intracellular Parasite Transcriptome of Enucleated Cells Hosting Leishmania amazonensis: Control of Half-Life of Host Cell Transcripts by the Parasite.

Enucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts that are more preserved or degraded upon interaction with parasites and also parasite genes that are differentially expressed when hosted by nucleated or enucleated cells. Cytoplasts are suitable host cells, which persist in culture for more than 72 h and display functional enrichment of transcripts related to mitochondrial functions and mRNA translation. Crosstalk between nucleated host de novo gene expression in response to intracellular parasitism and the parasite gene expression to counteract or benefit from these host responses induces a parasite transcriptional profile favoring parasite multiplication and aerobic respiration, and a host-parasite transcriptional landscape enriched in host cell metabolic functions related to NAD, fatty acid, and glycolytic metabolism. Conversely, interruption of host nucleus-parasite cross talk by infection of enucleated cells generates a host-parasite transcriptional landscape in which cytoplast transcripts are enriched in phagolysosome-related pathway, prosurvival, and SerpinB-mediated immunomodulation. In addition, predictive in silico analyses indicated that parasite transcript products secreted within cytoplasts interact with host transcript products conserving the host V-ATPase proton translocation function and glutamine/proline metabolism. The collective evidence indicates parasite-mediated control of host cell transcripts half-life that is beneficial to parasite intracellular multiplication and escape from host immune responses. These findings will contribute to improved drug targeting and serve as database for L. amazonensis-host cell interactions.

Insulin-Like Growth Factor-I as an Effector Element of the Cytokine IL-4 in the Development of a Leishmania major Infection.

Certain cytokines modulate the expression of insulin-like growth factor- (IGF-) I. Since IL-4 and IGF-I promote growth of the protozoan Leishmania major, we here addressed their interaction in downregulating the expression of Igf-I mRNA using small interfering RNA (siRNA) in Leishmania major-infected macrophages. Parasitism was decreased in the siRNA-treated cells compared with the nontreated cells, reversed by the addition of recombinant IGF-I (rIGF-I). In IL-4-stimulated macrophages, parasitism and the Igf-I mRNA amount were increased, and the effects were nullified upon siRNA transfection. IGF-I downregulation inhibited both parasite and macrophage arginase activation even in IL-4-stimulated cells. Searching for intracellular signaling components shared by IL-4 and IGF-I, upon siRNA transfection, phosphorylated p44, p38, and Akt proteins were decreased, affecting the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. In L. major-infected C57BL6-resistant mice, the preincubation of the parasite with rIGF-I changed the infection profile to be similar to that of susceptible mice. We conclude that IGF-I constitutes an effector element of IL-4 involving the PI3K/Akt pathway during L. major infection.

In vivo assessment of antiretroviral therapy-associated side effects.

Antiretroviral therapy has been associated with side effects, either from the drug itself or in conjunction with the effects of human immunodeficiency virus infection. Here, we evaluated the side effects of the protease inhibitor (PI) indinavir in hamsters consuming a normal or high-fat diet. Indinavir treatment increased the hamster death rate and resulted in an increase in triglyceride, cholesterol and glucose serum levels and a reduction in anti-oxLDL auto-antibodies. The treatment led to histopathological alterations of the kidney and the heart. These results suggest that hamsters are an interesting model for the study of the side effects of antiretroviral drugs, such as PIs.

Malaria in pregnant women living in areas of low transmission on the southeast Brazilian Coast: molecular diagnosis and humoural immunity profile.

Studies on autochthonous malaria in low-transmission areas in Brazil have acquired epidemiological relevance because they suggest continued transmission in what remains of the Atlantic Forest. In the southeastern portion of the state of São Paulo, outbreaks in the municipality of Juquitiba have been the focus of studies on the prevalence of Plasmodium, including asymptomatic cases. Data on the occurrence of the disease or the presence of antiplasmodial antibodies in pregnant women from this region have not previously been described. Although Plasmodium falciparum in pregnant women has been widely addressed in the literature, the interaction of Plasmodium vivax and Plasmodium malariae with this cohort has been poorly explored to date. We monitored the circulation of Plasmodium in pregnant women in health facilities located in Juquitiba using thick blood film and molecular protocols, as well as immunological assays, to evaluate humoural immune parameters. Through real-time and nested polymerase chain reaction, P. vivax and P. malariae were detected for the first time in pregnant women, with a positivity of 5.6%. Immunoassays revealed the presence of IgG antibodies: 44% for ELISA-Pv, 38.4% for SD-Bioline-Pv and 18.4% for indirect immunofluorescence assay-Pm. The high prevalence of antibodies showed significant exposure of this population to Plasmodium. In regions with similar profiles, testing for a malaria diagnosis might be indicated in prenatal care.

The bovine leukemia virus infection prolongs immunosuppression in dairy cows during the periparturient period by sustaining higher expression of immunological checkpoints in T cells.

Bovine leukemia virus (BLV) is caused by a deltaretrovirus and has been associated with immunosuppression as well as comorbidities such as bovine mastitis, the costliest disease in the dairy sector. However, no previous study has explored at the synergistic immunosuppressive effect of the peripartum period with an immunosuppressive viral disease such as BLV. Thus, our study explored the effect of BLV infection in the periparturient period on the expression of PD-1 and CTLA-4 in blood T lymphocytes, and the impact of BLV infection on the rate of new intramammary infections during the early lactation. Here, we found that BLV-infected dairy cows always had a statistically significant higher expression of CTLA-4 and PD-1 in blood T cells. Furthermore, our findings indicated that BLV infection prolongs immunosuppression in dairy cows during the periparturient period by sustaining higher expression of immunological checkpoints in T cells. In addition, BLV-infected dairy cows have a higher rate of new intramammary infections during early lactation. Thus, our study provides new insights of the immunosuppressive effect of BLV on the most critical period of the cows' life with marked detrimental effect on protective T-cell immunity and comorbidities, such as bovine mastitis.

Corrigendum: miR-548d-3p Is Up-regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages.

[This corrects the article DOI: 10.3389/fcimb.2022.826039.].

miR-548d-3p Is Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages.

Visceral leishmaniasis caused by Leishmania (Leishmania) infantum in Latin America progress with hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and weight loss and maybe lethal mainly in untreated cases. miRNAs are important regulators of immune and inflammatory gene expression, but their mechanisms of action and their relationship to pathogenesis in leishmaniasis are not well understood. In the present study, we sought to quantify changes in miRNAs associated with immune and inflammatory pathways using the L. (L.) infantum promastigote infected- human monocytic THP-1 cell model and plasma from patients with visceral leishmaniasis. We identified differentially expressed miRNAs in infected THP-1 cells compared with non-infected cells using qPCR arrays. These miRNAs were submitted to in silico analysis, revealing targets within functional pathways associated with TGF-ß, chemokines, glucose metabolism, inflammation, apoptosis, and cell signaling. In parallel, we identified differentially expressed miRNAs in active visceral leishmaniasis patient plasma compared with endemic healthy controls. In silico analysis of these data indicated different predicted targets within the TGF-ß, TLR4, IGF-I, chemokine, and HIF1α pathways. Only a small number of miRNAs were commonly identified in these two datasets, notably with miR-548d-3p being up-regulated in both conditions. To evaluate the potential biological role of miR-548d-3p, we transiently transfected a miR-548d-3p inhibitor into L. (L.) infantum infected-THP-1 cells, finding that inhibition of miR-548d-3p enhanced parasite growth, likely mediated through reduced levels of MCP-1/CCL2 and nitric oxide production. Further work will be required to determine how miR-548d-3p plays a role in vivo and whether it serves as a potential biomarker of progressive leishmaniasis.

Brief Research Report: Expression of PD-1 and CTLA-4 in T Lymphocytes and Their Relationship With the Periparturient Period and the Endometrial Cytology of Dairy Cows During the Postpartum Period.

The present study sought to evaluate the expression of PD-1 and CTLA-4 in blood T lymphocytes during the periparturient period and their relationship with uterine health in dairy cows, as determined by endometrial cytology and serum concentrations of ß-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs), which are indicators of a negative energy balance. The second objective of this study was to investigate whether the expression of PD-1 and CTLA-4 in T lymphocytes is associated with the serum acute phase-protein haptoglobin concentration during the periparturient period. To address these objectives, 26 clinically healthy dairy cows were used. Peripheral blood was collected 14 days prepartum (T-14), at calving (T0), and 30 days postpartum (T30) to measure the expression of PD-1 and CTLA-4 in blood T lymphocytes by flow cytometry. In addition, we collected blood at T0, 10 days after parturition (T10), and T30 to obtain serum and determine the serum concentrations of NEFA, BHB, and Hp. Endometrial cytology was performed at T10, 20 days after parturition (T20), and T30. In the present study, we observed higher expression of CTLA-4 and PD-1 in T lymphocytes at parturition and in the prepartum period, which could indicate a relationship between these immune checkpoints and immunological tolerance during gestation in dairy cattle. In addition, a negative association between the expression of these immune checkpoints prepartum or at parturition and endometrial cytology at T20 and T30 was observed, indicating the negative implications of these immune response regulators in susceptibility to infections. This finding was further corroborated by the relationship between the serum concentration of haptoglobin and the expression of CTLA-4 and PD-1 by T lymphocytes. However, we did not observe a relationship between the indicators of negative energy balance, evaluated by the serum concentrations of BHB and NEFA, and the expression of the immune checkpoint markers studied. Thus, our findings represent an initial step that paves the way for the development of new therapeutic alternatives directed by the host with the objective of increasing the resistance of dairy cattle to infections in this critical period of life.