Evidence that the p38 MAP kinase pathway is dysregulated in HLA–B27–expressing human monocytic cells: Correlation with HLA–B27 misfolding

Abstract

Objective. To investigate the cause of the enhanced intracellular replication of Salmonella enteritidis in HLA-B27-transfected U937 human monocytic cells and the contribution of HLA-B27 heavy chain (HC) misfolding.Methods. U937 monocytic cell transfectants stably expressing pSV2neo resistant vector (mock), wildtype HLA-B27, or mutated HLA-B27 HCs with amino acid substitutions in the B pocket were differentiated, infected with S enteritidis, and treated with signaling pathway inhibitors or specific p38 small interfering RNA (siRNA). The numbers of living intracellular bacteria were determined with the colony-forming unit method. To visualize S enteritidis, the bacteria were transformed with green fluorescent protein, and studied by microscopy.Results. Treatment with the p38 MAPK inhibitors or with p38 siRNA enhanced the replication of S enteritidis in U937 transfectants, whereas the other inhibitors had no effect. In mock-transfected cells and in cells expressing the mutated B27 HCs in which the misfolding had been corrected, p38 inhibitors impaired their ability to resist the replication of bacteria (mock, B27.A2B, B27.E45M, and B27.C67A). In contrast, the number of intracellular bacteria was not significantly increased in p38 inhibitor-treated cells expressing misfolded B27 HCs (B27g, B27cDNA, and B27.H9F).Conclusion. Our results show that p38 activity plays a crucial role in controlling intracellular S enteritidis in U937 cells. Enhanced replication of bacteria in B27-expressing cells requires that the HCs contain glutamic acid at position 45 and cysteine at position 67. Furthermore, in transfectants expressing misfolded B27 HCs, p38 inhibition had no significant effect on bacterial replication, suggesting that in these cells, the p38 pathway may not function properly.

HLA-B27 Modulates Intracellular Growth of Salmonella Pathogenicity Island 2 Mutants and Production of Cytokines in Infected Monocytic U937 Cells

“…Previously, several different theories were proposed to explain the role of HLA-B27 in the pathogenesis of ReA [4], [34]. Our earlier studies showed evidence that HLA-B27 influences the intracellular persistence of arthritis-triggering Salmonella within monocytes [6], [10]. These results support the hypothesis of ineffective elimination of microbes in the pathogenesis of ReA [35].…”

“…The expression of transfected HLA-B27 and HLA-A2 was confirmed by flow cytometry always in new batches of the cells as before [10], [11]. The level of expression of the transfected molecules in U937 cells was similar to that of HLA-B51, one of the MHC class I molecules endogenously expressed by U937 cells.…”

HLA-B27 and Human β2-Microglobulin Affect the Gut Microbiota of Transgenic Rats

“…Subsequent studies showed that mono-association of HLA-B27 transgenic rats with Bacteroides vulgatus in a germ-free environment was sufficient to re-establish colitis in these animals [22], [23]. A series of studies by Granfors et al have demonstrated marked effects of HLA-B27 on innate immunity and host defense in vitro , including effects on bacterial invasion of cells, intracellular persistence, intracellular signaling, and cytokine production [24]–[28]. Penttinen et al showed that HLA-B27-transfected monocytes had enhanced inflammatory responses to lipopolysaccharide found on bacterial cell walls [26], [29].…”

HLA-B27 Misfolding and Spondyloarthropathies

“…Recent evidence suggests that bacterial replication is increased 38 and that the p38 MAP kinase pathway may be disrupted. 39 This most likely represents an intracellular or at least nonantigen-presenting effect of HLA-B27. 40 Experiments using site-directed mutants of HLA-B27 show that the biological effect correlates with heavy chain misfolding, but is not associated with acute UPR activation and therefore the molecular mechanism remains to be defined.…”