SPI-1-encoded type III secretion system of Salmonella enterica is required for the suppression of porcine alveolar macrophage cytokine expression

Abstract

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.

Figure 1

Invasion and cytotoxicity of S. Typhimurium and S. Enteritidis for porcine alveolar macrophages. Panel A, invasion of the wild-type S. Typhimurium (wt) and isogenic ΔSPI1 mutant, and S. Enteritidis (wt) and isogenic ΔSPI1, sipA, hilA and sipB mutants into porcine alveolar macrophages 4 h post infection. Panel B, cytotoxicity of S. Typhimurium, S. Enteritidis and their isogenic mutants for porcine alveolar macrophage 24 h post infection. Controls included LDH released from non-infected PAMs, PAMS exposed to LPS, tissue culture medium, and PAMs lysed with 1% Triton X-100. * significantly different at p < 0.05 when t-test compared with appropriate wild-type strain.

Figure 2

ELISA detection of IL-1β, IL-8 and TNFα in tissue culture supernatants of non-stimulated PAMs and those exposed for 24 h to S. Typhimurium, ΔSPI1 mutant and LPS (1 μg/mL). *significantly different at p < 0.05 when t-test compared with the wild-type strain.

Figure 3

Cytokine expression of PAMs infected with the wild-type S. Enteritidis, sipA, hilA, ΔSPI1 and sipB mutants, and non-infected cells (n.i.).