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first_page settings Order Article Reprints Open AccessArticle Heterogeneity of Phenotypic and Functional Changes to Porcine Monocyte-Derived Macrophages Triggered by Diverse Polarizing Factors In Vitro by Giulia Franzoni 1,*, Lorena Mura 1,2, Elisabetta Razzuoli 3, Chiara Grazia De Ciucis 3, Floriana Fruscione 3, Filippo Dell’Anno 3, Susanna Zinellu 1, Tania Carta 1,4, Antonio G. Anfossi 4, Silvia Dei Giudici 1, Simon P. Graham 5and Annalisa Oggiano 1 1 Department of Animal Health, Istituto Zooprofilattico Sperimentale della Sardegna, 07100 Sassari, Italy 2 Department of Biomedical Sciences, School of Medicine, University of Sassari, 07100 Sassari, Italy 3 National Reference Center of Veterinary and Comparative Oncology (CEROVEC), Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta, 16129 Genova, Italy 4 Department of Veterinary Medicine, University of Sassari, 07100 Sassari, Italy 5 The Pirbright Institute, Ash Road, Pirbright, Woking GU24 ONF, UK * Author to whom correspondence should be addressed. Int. J. Mol. Sci.2023,24(5), 4671;https://doi.org/10.3390/ijms24054671 Received: 27 January 2023/Revised: 23 February 2023/Accepted: 25 February 2023/Published: 28 February 2023 (This article belongs to the Special IssueMacrophage Polarization: Learning to Manage It 2.0) Download Browse Figures ReviewReportsVersionsNotes Abstract Swine are attracting increasing attention as a biomedical model, due to many immunological similarities with humans. However, porcine macrophage polarization has not been extensively analyzed. Therefore, we investigated porcine monocyte-derived macrophages (moMΦ) triggered by either IFN-γ + LPS (classical activation) or by diverse “M2-related” polarizing factors: IL-4, IL-10, TGF-β, and dexamethasone. IFN-γ and LPS polarized moMΦ toward a proinflammatory phenotype, although a significant IL-1Ra response was observed. Exposure to IL-4, IL-10, TGF-β, and dexamethasone gave rise to four distinct phenotypes, all antithetic to IFN-γ and LPS. Some peculiarities were observed: IL-4 and IL-10 both enhanced expression of IL-18, and none of the “M2-related” stimuli induced IL-10 expression. Exposures to TGF-β and dexamethasone were characterized by enhanced levels of TGF-β2, whereas stimulation with dexamethasone, but not TGF-β2, triggered CD163 upregulation and induction of CCL23. Macrophages stimulated with IL-10, TGF-β, or dexamethasone presented decreased abilities to release proinflammatory cytokines in response to TLR2 or TLR3 ligands: IL-10 showed a powerful inhibitory activity for CXCL8 and TNF release, whereas TGF-β provided a strong inhibitory signal for IL-6 production. While our results emphasized porcine macrophage plasticity broadly comparable to human and murine macrophages, they also highlighted some peculiarities in this species.

34 Pág., African swine fever (ASF) is a hemorrhagic viral disease of domestic pigs and wild suids (all Sus scrofa) caused by the ASF virus (ASFV). The disease is spreading worldwide without control, threatening pig production due to the absence of licensed vaccine or commercially available treatments. A thorough understanding of the immunopathogenic mechanisms behind ASFV infection is required to better fight the disease. Cytokines are small, non-structural proteins, which play a crucial role in many aspects of the immune responses to viruses, including ASFV. Infection with virulent ASFV isolates often results in exacerbated immune responses, with increased levels of serum pro-inflammatory interleukins (IL-1α, IL-1β, IL-6), TNF and chemokines (CCL2, CCL5, CXCL10). Increased levels of IL-1, IL-6 and TNF are often detected in several tissues during acute ASFV infections and associated with lymphoid depletion, hemorrhages and oedemas. IL-1Ra is frequently released during ASFV infection to block further IL-1 activity, with its implication in ASFV immunopathology having been suggested. Increased levels of IFN-α and of the anti-inflammatory IL-10 seem to be negatively correlated with animal survival, whereas some correlation between virus-specific IFN-γ-producing cells and protection has been suggested in different studies where different vaccine candidates were tested, although future works should elucidate whether IFN-γ release by specific cell types is related to protection or disease development., Authors would like to thank the Italian Ministry of Health (grant IZS SA 06/21) for funding. Authors would like to thank the support received from the European project VACDIVA (EU-H2020-862874).

Cadmium (Cd2+) is regarded as one of the most toxic heavy metals, which can enter the food chain through environmental contamination and be bioaccumulated. Its exposure in Ligurian wild boars was monitored between 2016-2020 and revealed high level of this heavy metal in different provinces. In one of these polluted area, 21 wild boars were additionally sampled and the relationship between hepatic and renal Cd2+concentration suggested that majority of these animals presented chronic intoxication. Cd2+exposure of wild boar might lead to an immunosuppression status, thusin vitroexperiments on wild boar monocyte-derived macrophages (moMФ) were carried out. Effects of Cd2+scalar doses were evaluated through viability and adsorption assays, ELISA, qPCR. Moderate doses of this environmental pollutant (20 μM) were absorbed by moMФ, with subsequent reduction of their viability. This heavy metal did not trigger release of either IFN- β, anti-inflammatory or pro-inflammatory cytokines by moMФ, instead 24 h treatment with 20 μM of Cd2+resulted in down-regulated expression of TNF-α, IL-12p40, several TLRs, CD14, MD2, BD2, MyD88, p65, and NOS2. The results of our monitoring activity suggested that wild boar can be useful to monitor environmental exposure of this heavy metal and can help in understanding the type of contamination. In addition,in vitroexperiments on wild boar moMФ revealed that Cd2+exposure negatively affected the immune function of these cells, likely leading to increased susceptibility to infection.

Simple Summary Macrophages play a central role in innate immune response to both infectious and non-infectious stressors. They respond to different agonists modifying their phenotype and functions. In humans and mice, the regulatory cytokines IL-10 or TGF-β are both known to drive macrophage polarization into an anti-inflammatory phenotype, referred to as M2c. However, the immune systems of animal species each have their own peculiarities and the M2c subsets has never been investigated in pigs. A deep knowledge of the porcine immune system is required to design vaccines or control strategies against pathogens, which are a major constraint to pork production. Due to anatomical, physiological, and immunological similarities, swine are attracting increasing attention as a model for human diseases. To better characterize porcine macrophages, we evaluated the effects of IL-10 or TGF-β on the phenotype and function of monocyte-derived macrophages. Both cytokines downregulated the expression of MHC II DR and CD14. IL-10, but not TGF-β, statistically significantly reduced the ability of macrophages to respond to Toll-like receptor 2 (TLR2) or TLR4 agonists. Whilst these data suggest differentiation to an M2c-like immunosuppressive phenotype, the responses, and differences between IL-10 and TGF-β also reveals species-specific differences. Abstract Macrophages are phagocytic cells involved in maintaining tissue homeostasis and defense against pathogens. Macrophages may be polarized into different functionally specialized subsets. M2c macrophages arise following stimulation with IL-10 or TGF-β and mediate anti-inflammatory and tissue repair functions. M2c macrophages remain poorly characterized in the pig, thus we investigated the impact of these regulatory cytokines on porcine monocyte-derived macrophages (moMΦ). The phenotype and functionality of these cells was characterized though confocal microscopy, flow cytometry, ELISA, and RT-qPCR. Both cytokines induced CD14 and MHC II DR down-regulation and reduced IL-6, TNF-α, and CD14 expression, suggestive of an anti-inflammatory phenotype. Interestingly, neither IL-10 or TGF-β were able to trigger IL-10 induction or release by moMΦ. Differences between these cytokines were observed: stimulation with IL-10, but not TGF-β, induced up-regulation of both CD16 and CD163 on moMΦ. In addition, IL-10 down-regulated expression of IL-1β and IL-12p40 4h post-stimulation and induced a stronger impairment of moMΦ ability to respond to either TLR2 or TLR4 agonists. Overall, our results provide an overview of porcine macrophage polarization by two immunosuppressive cytokines, revealing differences between IL-10 and TGF-β, and reporting some peculiarity of swine, which should be considered in translational studies.

African swine fever (ASF) is a contagious viral disease of wild and domestic pigs that is present in many parts of Africa, Asia and Europe, including Sardinia (Italy). Deletions in the EP402R and B602L genes have been found in almost all ASF virus (ASFV) strains circulating in Sardinia from 1990 onwards, and modern Sardinian strains (isolated after 1990) might have acquired some selective advantage compared to historical ones (isolated before 1990). Here, we analysed the host cell responses of wild boars and domestic pigs upon infection with virus variants. Higher intracellular levels of the late protein p72 were detected after infection with the modern strain 22653/14 compared to the historical strain Nu81.2, although both isolates grew at the same rate in both monocytes and monocyte-derived macrophages. Higher cytokine levels in the supernatants of ASFV-infected pig monocytes compared to pig macrophages and wild-boar cells were detected, with no differences between isolates.

African swine fever (ASF) is a devastating viral disease of domestic pigs and wild boar for which there is no vaccine available. The aetiological agent ASF virus (ASFV) has a predilection for cells of the myeloid lineage. Macrophages provide a first line defence against pathogens and are the main target of ASFV, thus several studies analysed their response to infection in terms of cytokine/chemokine expression and modulation of functionality. These studies have typically used macrophages differentiated in vitro from blood or bone marrow progenitors and few studies have focused on responses of polarized macrophages (M1, M2) or functional macrophage subsets isolated from different tissues. ASFV can also infect dendritic cells (DC), but regardless of their central role in the induction of adaptive immune responses, their role in ASFV infection was only partially analysed. Future studies on ASFV-DC interaction are needed, which should take into consideration the heterogeneity within this family, composed of different subsets whose phenotype is also organ specific. Other porcine immune cells such as γδ-T cells, NK cells and fibrocytes, can act as 'non-conventional' antigen-presenting cells (APCs). In particular, γδ-T cells from ASFV immune pigs were shown to present viral antigens to T cells, but no studies have further explored the interaction of ASFV with this cell type or other non-conventional APCs. In this review we will provide an overview of the interaction of APCs with ASFV, describing the differences between virulent and attenuated strains, and suggesting areas for possible future studies.

African swine fever (ASF) is a devastating disease for which there is no vaccine. The ASF virus (ASFV) can infect dendritic cell (DC), but despite the critical role these cells play in induction of adaptive immunity, few studies investigated their response to ASFV infection. We characterized the in vitro interactions of porcine monocyte-derived DCs (moDC) with a virulent (22653/14), a low virulent (NH/P68) and an avirulent (BA71V) ASFV strain. At a high multiplicity of infection (MOI = 1), all three strains infected immature moDC. Maturation of moDC, with IFN-α/TNF-α, increased susceptibility to infection with 22653/14 and other virulent strains, but reduced susceptibility to NH/P68 and BA71V. The reduced moDC susceptibility to BA71V/NH/P68 was IFN-α mediated, whereas increased susceptibility to 22653/14 was induced by TNF-α. Using an MOI of 0.01, we observed that BA71V replicated less efficiently in moDC compared to the other isolates and we detected increased replication of NH/P68 compared to 22653/14. We observed that BA71V and NH/P68, but not 22653/14, downregulated expression of MHC class I on infected cells. All three strains decreased CD16 expression on moDC, whereas ASFV infection resulted in CD80/86 down-regulation and MHC class II DR up-regulation on mature moDC. None of the tested strains induced a strong cytokine response to ASFV and only modest IL-1α was released after BA71V infection. Overall our results revealed differences between strains and suggest that ASFV has evolved mechanisms to replicate covertly in inflammatory DC, which likely impairs the induction of an effective immune response.

Gut is often subject to infection by different pathogens like Y. enterocolitica. To date, biotypes (BTs) 1A have been considered as non-pathogenic, because they do not express plasmid of virulence pYV; however, BTs 1A strains present other chromosomic virulence genes and recent studies suggest an implication of this microorganism in reactive arthritis. Although many studies highlighted the molecular basis of pathogenesis of Ye infection, scanty data are available about several environmental BTs 1A strains, often isolated in cases of foodborne disease but not included in pathogenicity studies. The aim of our work was to verify the ability of different Ye 1A strains to adhere and penetrate IPEC-J2 cells and to modulate intestinal innate immunity. Our results showed that all strains under study were able to adhere and penetrate enterocytes, causing inflammatory responses. Indeed, adhesion and invasion of enterocytes is an essential step in Ye pathogenesis (Fàbrega and Vila, 2012). Moreover, our data suggest the possible involvement of strains Ye2/O:9 in reactive arthritis, due to their ability (i) to penetrate enterocytes as pathogenic Ye1/O:8 strains do, and (ii) to increase IL-6, IL-8, IL-12 and IL-18 release. Lastly, our results confirm that IPEC-J2 cells are a very good model to evaluate host-pathogen interaction, and indicate IL-8, TNF-α, TLRs1 and 4 as possible markers of the ability of Ye strains to penetrate enterocytes. Moreover, we showed that Ye strains differently affect the host's innate immune responses.

African swine fever (ASF) is a devastating disease for which there is no vaccine available. The ASF virus (ASFV) primarily infects cells of the myeloid lineage and this tropism is thought to be crucial for disease pathogenesis. A detailed in vitro characterization of the interactions of a virulent Sardinian isolate (22653/14) and a tissue culture adapted avirulent strain (BA71V) of ASFV with porcine monocytes, un-activated (moMΦ), classically (moM1) and alternatively (moM2) activated monocyte-derived macrophages was conducted in an attempt to better understand this relationship. Using a multiplicity-of-infection (MOI) of 1, both viruses were able to infect monocytes and macrophage subsets, but BA71V presented a reduced ability to infect moM1 compared to 22653/14, with higher expression of early compared to late proteins. Using an MOI of 0.01, only 22653/14 was able to replicate in all the macrophage subsets, with initially lowest in moM1 and moM2. No differences were observed in the expression of CD163 between ASFV infected and uninfected bystander cells. ASFV down-regulated CD16 expression but did not modulate MHC class II levels in monocytes and macrophage subsets. BA71V-infected but not 22653/14-infected moMΦ and moM2 presented with a reduced expression of MHC class I compared to the mock-infected controls. Higher levels of IL-18, IL1-β and IL-1α were released from moM1 after infection with BA71V compared to 22653/14 or mock-infected control. These results revealed differences between these ASFV strains, suggesting that virulent isolates have evolved mechanisms to counteract activated macrophages responses, promoting their survival, dissemination in the host and so ASF pathogenesis.