Immunomodulation by vasoactive intestinal peptide is associated with increased survival and growth of Salmonella Typhimurium in mice.

Studies have shown that administration of vasoactive intestinal peptide (VIP) in mice rescues them from lethal endotoxaemia and that this is correlated with decreased concentration of inflammatory cytokines. VIP has, therefore, been proposed as a novel anti-inflammatory which could be used in the treatment of Gram negative sepsis. However, the effect of VIP has not been reported in mice infected with viable Gram negative bacteria. Here, we show that Salmonella enterica serovar Typhimurium 4/74 significantly increased expression of mRNA of a type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in murine ileum and mesenteric lymph nodes at day 6 post-infection (d6 pi) and in the spleen at d3 pi. When VIP (5 nmol/ml) was administered to S. Typhimurium-infected mice, there was a significant increase in the number of S. Typhimurium cultured from murine faeces and ileum at d3 and 6 pi and in MLN and spleen at d3 dpi, compared to faeces and tissues examined from mice infected with S. Typhimurium (without VIP administration). Administration of VIP to S. Typhimurium-infected mice also altered the splenic architecture, resulting in a lack of discernable periarterial lymphoid sheaths or marginal zones at d6 pi but liver histology appeared similar on both d3 and d6 pi. The effects of VIP administration were correlated with a significant decrease in expression of inflammatory cytokine mRNA, associated with systemic inflammatory response syndrome (SIRS) of bacteraemia and acute sepsis. We conclude that VIP inhibits expression of diagnostic/prognostic cytokine biomarkers of sepsis in S. Typhimurium-infected mice. However, this occurred with a concomitant increase in Salmonella growth in tissues and increased bacterial shedding in faeces. Thus, VIP may have potential as an adjunctive therapy to antibiotics in sepsis.

Immune evasion by Salmonella: exploiting the VPAC1/VIP axis in human monocytes.

Immune evasion is a critical survival mechanism for bacterial colonization of deeper tissues and may lead to life-threatening conditions such as endotoxaemia and sepsis. Understanding these immune evasion pathways would be an important step for the development of novel anti-microbial therapeutics. Here, we report a hitherto unknown mechanism by which Salmonella exploits an anti-inflammatory pathway in human immune cells to obtain survival advantage. We show that Salmonella enterica serovar Typhimurium strain 4/74 significantly (P < 0·05) increased expression of mRNA and surface protein of the type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in human monocytes. However, we also show that S. Typhimurium induced retrograde recycling of VPAC1 from early endosomes to Rab11a-containing sorting endosomes, associated with the Golgi apparatus, and anterograde trafficking via Rab3a and calmodulin 1. Expression of Rab3a and calmodulin 1 were significantly increased by S. Typhimurium infection and W-7 (calmodulin antagonist) decreased VPAC1 expression on the cell membrane while CALP-1 (calmodulin agonist) increased VPAC1 expression (P < 0·05). When infected monocytes were co-cultured with VIP, a significantly higher number of S. Typhimurium were recovered from these monocytes, compared with S. Typhimurium recovered from monocytes cultured only in cell media. We conclude that S. Typhimurium infection exploits host VPAC1/VIP to gain survival advantage in human monocytes.

Differential Immune Phenotypes in Human Monocytes Induced by Non-Host-Adapted Salmonella enterica Serovar Choleraesuis and Host-Adapted S. Typhimurium.

We studied the effects of two Salmonella enterica serovar Typhimurium (host-adapted) strains (14028 and 4/74) and three S Choleraesuis (non-host-adapted) strains (A50, A45, and B195) in human monocytes between 2 and 24 h postinfection (p.i.) to investigate whether differences in immune response may explain the much higher prevalence of sepsis in individuals infected with S Choleraesuis. Both serovars significantly increased the production of cytokines associated with acute sepsis (tumor necrosis factor alpha [TNF-α], interleukin ß [IL-ß], and IL-6), but temporal differences occurred between these serovars and between different S Choleraesuis strains. Generally, all S Choleraesuis strains induced significantly higher production of inflammatory cytokines than S Typhimurium strains (P < 0.01 to 0.05). All S Choleraesuis strains very significantly increased IL-10 production by monocytes at 6 and 24 h p.i. in comparison to S Typhimurium strains (P < 0.01). In addition, ∼80% of monocytes were viable at 24 h p.i. with S Choleraesuis A50, compared to only ∼40% following S Typhimurium infection. Using S Typhimurium 14028 and S Choleraesuis A50 as examples of these two serovars, we also showed differential expression of genes within the Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) (JAK/STAT) pathway via quantitative PCR (qPCR) microarray analysis. High serum IL-10 concentration and monocyte survival have been reported as markers of the development of human sepsis. We therefore conclude that high production of IL-10 by monocytes may, in part, explain the greater propensity for S Choleraesuis to induce human sepsis and that this may be greater in strains such as A50, which induces both high IL-10 production and monocyte survival.

Dysregulation of JAK/STAT genes by vasoactive intestinal peptide (VIP) in Salmonella-infected monocytes may inhibit its therapeutic potential in human sepsis.

Murine/LPS models of Gram negative sepsis indicate that vasoactive intestinal peptide (VIP) has therapeutic potential. We investigated the unknown effect of VIP on JAK/STAT proteins and genes in human monocytes infected with Salmonella Typhimurium 14028. S. Typhimurium 14028 increased expression of both IL-6 receptor (IL-6R) and interferon gamma receptor 1 (IFNγR1) on monocytes but co-culture of infected monocytes with VIP (10-7 M) only decreased expression of IFNγR1 (P < 0.05). In contrast, S. Typhimurium 14028 infection or co-culture with VIP had no effect on IL-10 receptor expression on the monocyte surface. However, S. Typhimurium 14028 down regulated IFNGR1 gene expression and this was not altered by co-culture with VIP, suggesting that changes in IFNγR1 protein may be due to an effect on cytoplasmic transport. 15 JAK/STAT genes, out of 84 studied, were up-regulated by S. Typhimurium 14028 infection and five were down-regulated. Co-culture with VIP significantly decreased expression of two genes (IFNG and IL-20) and increased expression of three genes (SOCS1, SOCS3 and STAT4) (P < 0.05). S. Typhimurium 14028 also increased expression of PTPN1, which dephosphorylates JAK2 and TYK2. This was unaltered by co-culture with VIP but S. Typhimurium 14028-induced expression of ISG15, associated with susceptibility to Gram negative infection, was further increased by VIP. We conclude that the effect of VIP on JAK/STAT genes may preclude its therapeutic use in human Gram negative sepsis.

Vasoactive intestinal peptide (VIP) differentially affects inflammatory immune responses in human monocytes infected with viable Salmonella or stimulated with LPS.

We compared the effect of VIP on human blood monocytes infected with Salmonella typhimurium 4/74 or stimulated with LPS. VIP (10(-7)M) increased monocyte viability by 24% and 9% when cultured for 24h with 4/74 or Salmonella LPS (100ng/ml), respectively. Significantly increased (P<0.05) numbers of 4/74 were also recovered from monocytes co-cultured with VIP after 6h post-infection (pi) and this remained high after 24h pi. Both 4/74 and LPS increased (P<0.05) the concentration of TNF-α, IL-1ß and IL-6 measured in monocyte supernatants. However, LPS induced this effect more rapidly while, with the exception of IL-6, 4/74 induced higher concentrations (P<0.05). VIP significantly decreased (P<0.05) TNF-α and IL-1ß production by 4/74-infected monocytes after 6 pi, but only after 24h in LPS-cultured monocytes. This trend was reversed for IL-6 production. However, TNF-α and IL-1ß production by 4/74-infected monocytes, cultured with VIP, still remained higher (P<0.05) than concentrations measured in supernatants cultured only with LPS. VIP also increased (P<0.05) production of anti-inflammatory IL-10 in both 4/74 and LPS cultures after 24h. We also show a differential effect of VIP on the expression of TNFα and IL-6 receptors, since VIP was only able to decreased expression in LPS-stimulated monocytes but not in 4/74-infected monocytes. In conclusion, we show a differential effect of VIP on human monocytes infected with virulent Salmonella or stimulated with LPS. Our study suggests that the use of VIP in bacteraemia and/or sepsis may be limited to an adjunctive therapy to antibiotic treatment.