Milk lymphocyte profile and macrophage functions: new insights into the immunity of the mammary gland in quarters infected with Corynebacterium bovis

Silva, Vitória M.; Souza, Marina Trevelin; Blagitz, Maiara Garcia; Souza, Fernando Nogueira de; Batista, Camila Freitas; Alves, Alexandre José; Fernandes, Artur C. C.; Sánchez, E.; Ramirez, Carla María Ordinola; Costa, Luciana da; Libera, Alice Maria Melville Paiva Della

doi:10.1186/s12917-021-02989-5

Cited by 9 publications

(3 citation statements)

References 42 publications

(66 reference statements)

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“…The impact of TLR priming on the phagocytosis activity of purified neutrophils was analyzed using flow cytometry. [ 24 ]. For this, heat-killed S. aureus (Pansorbin) was purchased from Calbiochem (Nottingham, UK) and labeled with fluoresceinisothiocyanate (FITC) according to the manufacturer’s protocol (fluoresceinisothiocyanate labeling kit, Sigma-Aldrich, St. Louis, MO, USA).…”

Section: Animals Materials and Methodsmentioning

confidence: 99%

Impact of Selected Bacterial and Viral Toll-like Receptor Agonists on the Phenotype and Function of Camel Blood Neutrophils

Hussen

Alkuwayti

Falemban

et al. 2023

Veterinary Sciences

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Innate recognition of pathogens depends on the interaction between microbial structures known as pathogen-associated molecular patterns (PAMPs) and pattern recognition receptors (PRRs) in host cells. Toll-like receptors (TLR) are among the most important PRRs being expressed on and in a wide range of immune cell types. Studies on the interaction mechanisms between different pathogen species and the immune system of the dromedary camel are still scarce. The present study aimed to investigate the immunomodulatory effect of synthetic bacterial and viral TLR ligands on some phenotypic properties and selected functions of neutrophils purified from dromedary camel blood. Neutrophils were separated from camel blood (n = five animals) and were stimulated in vitro with the TLR ligands LPS, Pam3CSK4, R848 (Resiquimod), and Poly IC or were left without stimulation. Stimulation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) was used as a positive control stimulation. Shape change, phagocytosis activity, ROS production, the expression of cell surface markers, and cell vitality were compared between stimulated and non-stimulated cells. With exception of the TLR3 agonist Poly IC, all TLR ligands used showed the potential to stimulate camel neutrophils resulting in increased cell size and the upregulation of CD18 and CD14 on their surface. Similarly, the phagocytosis activity of camel neutrophils was significantly improved after priming with all TLR ligands, except Poly IC, which, in contrast, resulted in a reduced percentage of phagocytosis-positive cells. In contrast to stimulation with PMA, which induced a significant ROS production in camel neutrophils, none of the TLR ligands used stimulated ROS generation in neutrophils. Only stimulation with Pam3CSK4 increased the expression of MHCII molecules on camel neutrophils, resulting in an expanded MHCIIhigh fraction within camel neutrophils. Our study indicates selective immunomodulating effects of TLR agonists on purified camel neutrophils without affecting their vitality.

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Section: Animals Materials and Methodsmentioning

confidence: 99%

Impact of Selected Bacterial and Viral Toll-like Receptor Agonists on the Phenotype and Function of Camel Blood Neutrophils

Hussen

Alkuwayti

Falemban

et al. 2023

Veterinary Sciences

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“…Bacterial phagocytosis by milk phagocytes was performed using heat-killed staphylococcus aureus ( S. aureus ) bacteria (Pansorbin, Calbiochem, Merck, Nottingham, UK) labeled with fluoresceinisothiocyanate (FITC, Sigma-Aldrich, St. Louis, Missouri, USA) ( 54 ). Separated milk cells (2 × 10 5 in 100 μl RPMI medium) were incubated with S. aureus-FITC (50 bacteria/cell) for 45 min at 37°C in 96 well plates.…”

Section: Methodsmentioning

confidence: 99%

Milk Immune Cell Composition in Dromedary Camels With Subclinical Mastitis

et al. 2022

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Mastitis represents one of the most important infectious diseases in camels with heavy economic losses due to reduced milk quantity and quality. Balanced immune cell composition and function in the mammary gland are essential for effective immune response to mastitis pathogens. The objective of the present study was to characterize the cellular immune response to subclinical mastitis in the mammary gland of dromedary camels. Therefore, immunostaining and flow cytometry were used to compare the cellular composition, leukocyte phenotype, and cell viability in camel milk from healthy she-camels (n = 8) and she-camels with subclinical mastitis (SCM; n = 6). In addition, the ex vivo phagocytic activity of milk phagocytes was compared between healthy and affected animals. The health status of the mammary gland was evaluated based on the California Mastitis Test (CMT) score. SCM (CMT score of ≥3 in the absence of clinical signs of mastitis) was found in six of the 56 sampled quarters (10.7 %) with only one affected quarter per animal. In comparison to milk from healthy camels, milk from SCM animals showed higher somatic cell count (SCC), higher numbers of CD45+ leukocytes with an expanded fraction of CD172a+ myeloid cells. Within the myeloid cell population, there was an increase in the percentage of granulocytes (CD172a+CD14low) with a decreased percentage of macrophages (CD172a+CD14high) in milk from affected animals compared to healthy animals. The decrease in lymphoid cells in SCM milk was mainly due to the decreased fraction of CD4+ helper T cells. Camel SCM was also associated with a stimulated phenotype, increased cell viability, and enhanced phagocytic activity of the milk phagocytes, macrophages and granulocytes. Collectively, the present study identified significant changes in SCC, leukocyte count, phenotype, viability, and function in association with subclinical mastitis in camels. The results of the present study support a better understanding of host-pathogen interaction mechanisms in the camel mammary gland.

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“…The bacterial phagocytosis by camel MDM was performed by incubating MDM with heat-killed Staphylococcus aureus (S. aureus) bacteria (Calbiochem, Nottingham, UK) labeled with a FITC labeling kit (Sigma-Aldrich, Missouri, USA) according to the manufacturer instructions (Silva et al, 2021). Camel MDM (1 x 10 3 cells in 100 µL RPMI medium) were incubated with S. aureus-FITC (20 bacteria/cell) for 30 minutes at 37°C and 5% CO2.…”

Section: Phagocytosis and Reactive Oxygen Species Assaysmentioning

confidence: 99%

The Modulation of in Vitro Differentiation of Monocyte-derived Macrophage by Trypanosoma evansi Antigens in the Dromedary Camel

AL-Hilal,

Alkuwayti,

Alrabiah

et al. 2023

WVJ

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Studies on the camel immune response to Trypanosoma (T.) evansi, the causative agent of Surra, are very limited. In the present study, flow cytometry was employed to investigate the modulatory effects of different T. evansi antigens on the in vitro differentiation of camel blood monocytes into macrophages. For this, in vitro, separated camel monocytes were differentiated into monocyte-derived macrophages (MDM) in the presence or absence (control) of formalin-fixed (inactivated) T. evansi whole parasite (T. evansi group) or the purified Ro Tat 1.2 antigen (Ro Tat 1.2 group). The analysis of the antimicrobial functions of MDM (phagocytosis and reactive oxygen species (ROS) production) revealed reduced phagocytosis activity of camel MDM generated in the presence of T. evansi antigens. In addition, a lack of ROS-response was observed in camel MDM generated in the presence of T. evansi antigens after stimulation with PMA. These results indicated a compromising effect of T. evansi on the innate defense mechanisms in camels. Phenotypic analysis revealed the upregulation of major histocompatibility complex (MHC) class II molecules together with the lower abundance of the scavenger receptor for haptoglobin–hemoglobin complexes (CD163) on MDM generated in the presence of whole T. evansi parasites, indicating a polarizing effect of T. evansi on the differentiation of camel monocytes into an M1 phenotype. However, the reduced antimicrobial functions of these cells argue against their pro-inflammatory nature. Although both MDM generated in the presence of whole T. evansi antigens or their purified Ro Tat 1.2 proteins indicated similar expression levels of CD14 and MHCII molecules, the different abundance of the cell surface molecules CD172a, CD163, CD45, and CD44 indicated different phenotypes of the two MDMs. The results of the present study revealed compromising effects of T. evansi antigens on camel macrophages differentiated in vitro from blood monocytes. Whether these effects contribute to the in vivo pathogenesis of T. evansi in camels remains to be determined in future studies.

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Milk lymphocyte profile and macrophage functions: new insights into the immunity of the mammary gland in quarters infected with Corynebacterium bovis

Cited by 9 publications

References 42 publications

Impact of Selected Bacterial and Viral Toll-like Receptor Agonists on the Phenotype and Function of Camel Blood Neutrophils

Impact of Selected Bacterial and Viral Toll-like Receptor Agonists on the Phenotype and Function of Camel Blood Neutrophils

Milk Immune Cell Composition in Dromedary Camels With Subclinical Mastitis

The Modulation of in Vitro Differentiation of Monocyte-derived Macrophage by Trypanosoma evansi Antigens in the Dromedary Camel

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