Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release

Schnabel, Christiane L.; Steinig, P.; Schuberth, H. J.; Koy, Mirja; Wagner, Bettina; Wittig, Burghardt; Juhls, Christiane; Willenbrock, Saskia; Escobar, Hugo Murua; Jaehnig, Peter; Feige, Karsten; Cavalleri, Jessika‐M. V.

doi:10.1016/j.vetimm.2015.02.011

Cited by 12 publications

(13 citation statements)

References 75 publications

(93 reference statements)

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“…This could have arisen from the isolation and culture procedures themselves, from the presence of other TLR4 ligands such as host-derived heat-shock proteins [48], or from small quantities of contaminating LPS, which is ubiquitous in the horse’s environment. Spontaneous production of the cytokines studied here, of a comparable magnitude to the time-induced changes observed, have been previously reported in similar culture systems with equine PBMCs and are therefore not unexpected [49]. The differences in production of key LPS-induced cytokines and in miR-155 expression do however indicate that sufficient differential stimulation was achieved to observe an LPS effect.…”

Section: Discussionsupporting

confidence: 85%

Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing

et al. 2017

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The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.

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Section: Discussionsupporting

confidence: 85%

Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing

et al. 2017

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“…Mares showed higher increases in WBC ( t -test, p = 0.0068) and neutrophils ( t -test, p = 0.0282), and higher TNFα responses (medium settings; t -test, p = 0.0172) than geldings (Additional file 14 ). This is in accordance with the finding of higher plasma levels of TNFα in healthy mares [ 78 ] and the authors’ finding of a rather pro-inflammatory tendency of mares compared to geldings when analysing baseline measurements prior to treatments [ 79 ].…”

Section: Resultssupporting

confidence: 92%

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

Schnabel

Steinig

Koy³

et al. 2015

BMC Vet Res

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BackgroundDeoxyribonucleic acid (DNA) vaccines are used for experimental immunotherapy of equine melanoma. The injection of complexed linear DNA encoding interleukin (IL)-12/IL-18 induced partial tumour remission in a clinical study including 27 grey horses. To date, the detailed mechanism of the anti-tumour effect of this treatment is unknown.ResultsIn the present study, the clinical and cellular responses of 24 healthy horses were monitored over 72 h after simultaneous intradermal and intramuscular application of equine IL-12/IL-18 DNA (complexed with a transfection reagent) or comparative substances (transfection reagent only, nonsense DNA, nonsense DNA depleted of CG).Although the strongest effect was observed in horses treated with expressing DNA, horses in all groups treated with DNA showed systemic responses. In these horses treated with DNA, rectal temperatures were elevated after treatment and serum amyloid A increased. Total leukocyte and neutrophil counts increased, while lymphocyte numbers decreased. The secretion of tumour necrosis factor alpha (TNFα) and interferon gamma (IFNγ) from peripheral mononuclear blood cells ex vivo increased after treatments with DNA, while IL-10 secretion decreased. Horses treated with DNA had significantly higher myeloid cell numbers and chemokine (C-X-C motif) ligand (CXCL)-10 expression in skin samples at the intradermal injection sites compared to horses treated with transfection reagent only, suggesting an inflammatory response to DNA treatment.In horses treated with expressing DNA, however, local CXCL-10 expression was highest and immunohistochemistry revealed more intradermal IL-12-positive cells when compared to the other treatment groups.In contrast to non-grey horses, grey horses showed fewer effects of DNA treatments on blood lymphocyte counts, TNFα secretion and myeloid cell infiltration in the dermis.ConclusionTreatment with complexed linear DNA constructs induced an inflammatory response independent of the coding sequence and of CG motif content. Expressing IL-12/IL-18 DNA locally induces expression of the downstream mediator CXCL-10.The grey horses included appeared to display an attenuated immune response to DNA treatment, although grey horses bearing melanoma responded to this treatment with moderate tumour remission in a preceding study. Whether the different immunological reactivity compared to other horses may contributes to the melanoma susceptibility of grey horses remains to be elucidated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0452-3) contains supplementary material, which is available to authorized users.

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“…Likewise, Pam3CSK4 induces equine monocytes to produce IL-1β, IL-6, IL-10 and TNF-α [14] as it does in human and murine cells [25, 26]. In the present study, Pam3CSK4 did not induce gene expression of TNF-α, possibly explained by a spontaneous increase in gene expression of TNF-α seen after 18 h of incubation (data not shown) being congruent with a high spontaneous release of TNF-α found in supernatants of unstimulated eqPBMC [22]. Also, a spontaneous induction of IL-1β was recorded in the control cultures.…”

Section: Discussionsupporting

confidence: 83%

The adjuvant G3 promotes a Th1 polarizing innate immune response in equine PBMC

Hellman

Hjertner

Morein

et al. 2018

Vet Res

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The immunomodulatory effect of a new particulate adjuvant, G3, alone or in combination with agonists to TLR2/1 or TLR5 was evaluated in cultures of equine PBMC. Exposure to the G3 adjuvant up-regulated genes encoding IFN-γ, IL-1β, IL-6, IL-8, IL-12p40 and IL-23p19 in the majority of the horses tested, indicating that the G3 adjuvant induced a pro-inflammatory and Th1 dominated profile. In accordance, genes encoding IL-13, IL-4, IL-10 and TGF-β remained unaffected and genes encoding IFN-α, IL-17A and TNF-α were only occasionally and weakly induced. The two TLR agonists Pam3CSK4 (TLR2/1) and FliC (TLR5) induced cytokine profiles characterized by a clear induction of IL-10 as well as up-regulation of the genes encoding IL-1β, IL-6 and IL-8. The presence of G3 modified this response, in particular by reducing the FliC and Pam3CSK4 induced production of IL-10. Furthermore, G3 acted in synergy with Pam3CSK4 in enhancing the production of IFN-γ whereas G3 combined with FliC increased the gene expression of IL-8. Thus, the G3 adjuvant seems to have the capacity to promote a Th1 polarizing innate immune response in eqPBMC, both by favouring IFN-γ production and by reducing production of IL-10 induced by co-delivered molecules. These features make G3 an interesting candidate to further evaluate for its potential as an adjuvant in equine vaccines. Electronic supplementary material The online version of this article (10.1186/s13567-018-0602-2) contains supplementary material, which is available to authorized users.

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Influences of age and sex on leukocytes of healthy horses and their ex vivo cytokine release

Cited by 12 publications

References 75 publications

Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing

Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing

Immune response of healthy horses to DNA constructs formulated with a cationic lipid transfection reagent

The adjuvant G3 promotes a Th1 polarizing innate immune response in equine PBMC

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