Genes localized at Salmonella pathogenicity island-1 (SPI-1) are involved in Salmonella enterica invasion of host non-professional phagocytes. Interestingly, in macrophages, SPI-1-encoded proteins, in addition to invasion, induce cell death via activation of caspase-1 which also cleaves proIL-1β and proIL-18, precursors of 2 proinflammatory cytokines. In this study we were therefore interested in whether SPI-1-encoded type III secretion system (T3SS) may influence proinflammatory response of macrophages. To test this hypothesis, we infected primary porcine alveolar macrophages with wild-type S. Typhimurium and S. Enteritidis and their isogenic SPI-1 deletion mutants. ΔSPI1 mutants of both serovars invaded approx. 5 times less efficiently than the wild-type strains and despite this, macrophages responded to the infection with ΔSPI1 mutants by increased expression of proinflammatory cytokines IL-1β, IL-8, TNFα, IL-23α and GM-CSF. Identical macrophage responses to that induced by the ΔSPI1 mutants were also observed to the infection with sipB but not the sipA mutant. The hilA mutant exhibited an intermediate phenotype between the ΔSPI1 mutant and the wild-type S. Enteritidis. Our results showed that the SPI-1-encoded T3SS is required not only for cell invasion but in macrophages also for the suppression of early proinflammatory cytokine expression.

In this study we compared the proteomes of macrophages and heterophils isolated from the spleen 4 days after intravenous infection of chickens with Salmonella Enteritidis. Heterophils were characterized by expression of MMP9, MRP126, LECT2, CATHL1, CATHL2, CATHL3, LYG2, LYZ and RSFR. Macrophages specifically expressed receptor proteins, e.g. MRC1L, LRP1, LGALS1, LRPAP1 and a DMBT1L. Following infection, heterophils decreased ALB and FN1, and released MMP9 to enable their translocation to the site of infection. In addition, the endoplasmic reticulum proteins increased in heterophils which resulted in the release of granular proteins. Since transcription of genes encoding granular proteins did not decrease, these genes remained continuously transcribed and translated even after initial degranulation. Macrophages increased amounts of fatty acid elongation pathway proteins, lysosomal and phagosomal proteins. Macrophages were less responsive to acute infection than heterophils and an increase in proteins like CATHL1, CATHL2, RSFR, LECT2 and GAL1 in the absence of any change in their expression at RNA level could even be explained by capturing these proteins from the external environment into which these could have been released by heterophils.

Background: Within the last decade, macrophages have been shown to be capable of differentiating toward a classically activated phenotype (M1) with a high antimicrobial potential or an alternatively activated phenotype (M2). Some pathogens are capable of interfering with differentiation in order to down-regulate the anti-microbial activity and enhance their survival in the host. Results: To test this ability in Salmonella enterica serovar Typhimurium, we infected porcine alveolar macrophages with wild-type Salmonella Typhimurium and its isogenic mutants devoid of two major pathogenicity islands, SPI-1 and SPI-2. The induction of genes linked with M1 or M2 polarization was determined by quantification of gene expression by RT-qPCR. The ΔSPI-1 mutant induced a high, dose-dependent M1 response but a low M2 response in infected macrophages. On the other hand, wild-type Salmonella Typhimurium induced a low M1 response but a high, dose-dependent M2 response in infected macrophages. The response to ΔSPI-2 mutant infection was virtually the same as the wild-type strain. Conclusions: We therefore propose that Salmonella Typhimurium DT104 studied here can polarize macrophages towards the less bactericidal M2 phenotype and that this polarization is dependent on the type III secretion system encoded by SPI-1.

Following infection and initial multiplication in the gut lumen, Salmonella Typhimurium crosses the intestinal epithelial barrier and comes into contact with cells of the host immune system. Mononuclear phagocytes which comprise macrophages and dendritic cells (DC) are of key importance for the outcome of Salmonella infection. Although macrophages and DC may differentiate from a common precursor, their capacities to process and present antigen differ significantly. In this study, we therefore compared the response of porcine macrophages and DC differentiated from peripheral blood monocytes to S. Typhimurium and one of the most potent bacterial pathogen associated molecular patterns, bacterial lipopolysaccharide. To avoid any bias, the expression was determined by protein LC-MS/MS and verified at the level of transcription by quantitative RT-PCR. Within 4 days of culture, peripheral blood monocytes differentiated into two populations with distinct morphology and expression of MHC II. Mass spectrometry identified 446 proteins in macrophages and 672 in DC. Out of these, 433 proteins were inducible in macrophages either after infection with S. Typhimurium or LPS exposure and 144 proteins were inducible in DC. The expression of the 46 most inducible proteins was verified at the level of transcription and the differential expression was confirmed in 22 of them. Out of these, 16 genes were induced in both cell types, 3 genes (VCAM1, HMOX1 and Serglycin) were significantly induced in macrophages only and OLDLR1 and CDC42 were induced exclusively in DC. Thirteen out of 22 up-regulated genes contained the NF-kappaB binding site in their promoters and could be considered as either part of the NF-kappaB feedback loop (IkappaBalpha and ISG15) or as NF-kappaB targets (IL1beta, IL1alpha, AMCF2, IL8, SOD2, CD14, CD48, OPN, OLDLR1, HMOX1 and VCAM1). The difference in the response of monocyte derived macrophages and DC was quantitative rather than qualitative. Despite the similarity of the responses, compared to DC, the macrophages responded in a more pro-inflammatory fashion.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant and economically important infectious diseases affecting swine worldwide and can predispose pigs to secondary bacterial infections caused by, e.g. Haemophilus parasuis. The aim of the presented study was to compare susceptibility of two different types of macrophages which could be in contact with both pathogens during infection with PRRS virus (PRRSV) and in co-infection with H. parasuis. Alveolar macrophages (PAMs) as resident cells provide one of the first lines of defence against microbes invading lung tissue. On the other hand, monocyte derived macrophages (MDMs) represent inflammatory cells accumulating at the site of inflammation. While PAMs were relatively resistant to cytopathogenic effect caused by PRRSV, MDMs were much more sensitive to PRRSV infection. MDMs infected with PRRSV increased expression of pro-apoptotic Bad, Bax and p53 mRNA. Increased mortality of MDMs may be also related to a higher intensity of ROS production after infection with PRRSV. In addition, MDMs (but not PAMs) infected with H. parasuis alone formed multinucleated giant cells (MGC); these cells were not observed in MDMs infected with both pathogens. Higher sensitivity of MDMs to PRRSV infection, which is associated with limited MDMs survival and restriction of MGC formation, could contribute to the development of multifactorial respiratory disease of swine.

In attempt to identify genes that are induced in chickens by Salmonella Enteritidis we identified a new highly inducible gene, interleukin 4 induced 1 gene (IL4I1). IL4I1 reached its peak expression (458× induction) in the cecum of newly hatched chickens 4 days post-infection and remained upregulated for an additional 10 days. IL4I1 was expressed and induced in macrophages and granulocytes, both at the mRNA and protein level. IL4I1 was expressed and induced also in CD4 and γδ T-lymphocytes though at a 50-fold lower level than in phagocytes. Expression of IL4I1 was not detected in CD8 T lymphocytes or B lymphocytes. Mutation of IL4I1 in chicken HD11 macrophages did not affect their bactericidal capacity against S. Enteritidis but negatively affected their oxidative burst after PMA stimulation. We therefore propose that IL4I1 is not directly involved in bactericidal activity of phagocytes and, instead, it is likely involved in the control of inflammatory response and signaling to T and B lymphocytes.