Yersinia spp. undermine the immune responses of infected animals by translocating Yops directly into host cells with a type III secretion system. YopM, a leucine-rich repeat protein, is a critical virulence factor in infection. YopM localizes to both the nucleus and the cytoplasm in cultured cells, interacts with mammalian p90 ribosomal S6 kinase 1 (RSK1), and causes a decrease in NK cell populations in spleens. Little is known about the molecular interaction between YopM and RSK1 and its significance in pathogenesis. We performed a systematic deletion analysis of YopM in Yersinia pseudotuberculosis to determine which regions are required for RSK1 interactions, nuclear localization, virulence, and changes in immune cell populations during infection of mice. Full-length YopM associated with RSK1 in at least two protein complexes in infected cells, and deletion of its C-terminal tail abrogated all RSK1 interactions. The C-terminal tail was required for tissue colonization, as yopM mutants that failed to interact with RSK1 were as defective for tissue colonization as was a ⌬yopM mutant; however, nuclear localization of YopM was not dependent on its RSK1 interaction. Mutants expressing YopM proteins which do not interact with RSK1 caused more pathology than did the ⌬yopM mutant, suggesting that there are other RSK1-independent functions of YopM. Histopathological and flow cytometric analyses of spleens showed that infection with wild-type Y. pseudotuberculosis caused an influx of neutrophils, while mice infected with yopM mutants had increased numbers of macrophages. Decreases in NK cells after Y. pseudotuberculosis infection did not correlate with YopM expression. In conclusion, the C terminus of YopM is essential for RSK1 interactions and for virulence.

Hemophagocytic lymphohistiocytosis (HLH) is a hyper-inflammatory clinical syndrome associated with neoplastic disorders especially lymphoma, autoimmune conditions, and infectious agents including bacteria, viruses, protozoa and fungi. In both human and veterinary medicine, hemophagocytic histiocytic disorders are clinically important and frequently fatal. HLH in humans can be a primary (familial, autosomal recessive) or secondary (acquired) condition, with both types generally precipitated by an infectious agent. Previously, no mouse model for secondary HLH has been reported. Using Salmonella enterica serotype Typhimurium by oral gavage to mimic naturally-occurring infection in Sv129S6 mice, we characterized the clinical, hematologic and morphologic host responses to disease thereby describing an animal model with the clinico-pathologic features of secondary HLH as set forth by the Histiocyte Society: fever, splenomegaly, cytopenias (anemia, thrombocytopenia), hemophagocytosis in bone marrow and spleen, hyperferritinemia, and hypofibrinogenemia. Disease severity correlates with high splenic and hepatic bacterial load, and we show disease course can be monitored and tracked in live animals. Whereby secondary HLH is known to occur in human patients with typhoid fever and other infectious diseases, our characterization of a viable natural disease model of secondary HLH offers an important means to elucidate pathogenesis of poorly understood mechanisms of secondary HLH and investigation of novel therapies. We characterize previously unreported secondary HLH in a chronic mouse model of typhoid fever, and novel changes in hematology including decreased tissue ferric iron storage that differs from classically described anemia of chronic disease. Our studies demonstrate S. Typhimurium infection of mice is a natural infectious disease model of secondary HLH that may have utility for elucidating disease pathogenesis and developing novel therapies.

Salmonella enterica serovar Typhimurium (S. Typhimurium) causes systemic inflammatory disease in mice by colonizing cells of the mononuclear leukocyte lineage. Mouse strains resistant to S. Typhimurium, including Sv129S6, have an intact Nramp1 (Slc11a1) allele and survive acute infection, whereas C57/BL6 mice, homozygous for a mutant Nramp1 allele, Nramp1G169D, develop lethal infections. Restoration of Nramp1 (C57/BL6 Nramp1G169) reestablishes resistance to S. Typhimurium; mice survive at least 3 to 4 weeks postinfection. Since many transgenic mouse strains are on a C57/BL6 genetic background, C57/BL6 Nramp1G169 mice provide a model to examine host genetic determinants of resistance to infection. To further evaluate host immune response to S. Typhimurium, we performed comparative analyses of Sv129S6 and C57/BL6 Nramp1G169 mice 3 weeks following oral S. Typhimurium infection. C57/BL6 Nramp1G169 mice developed more severe inflammatory disease with splenic bacterial counts 1000-fold higher than Sv129S6 mice and relatively greater splenomegaly and blood neutrophil and monocyte counts. Infected C57/BL6 Nramp1G169 mice developed higher proinflammatory serum cytokine and chemokine responses (interferon-γ, tumor necrosis factor–α, interleukin [IL]–1β, and IL-2 and monocyte chemotactic protein–1 and chemokine [C-X-C motif] ligand 1, respectively) and marked decreases in anti-inflammatory serum cytokine concentrations (IL-10, IL-4) compared with Sv129S6 mice postinfection. Splenic dendritic cells and macrophages in infected compared with control mice increased to a greater extent in C57/BL6 Nramp1G169 mice than in Sv129S6 mice. Overall, data show that despite the Nramp1 gene present in both strains, C57/BL6 Nramp1G169 mice develop more severe, Th1-skewed, acute inflammatory responses to S. Typhimurium infection compared with Sv129S6 mice. Both strains are suitable model systems for studying inflammation in the context of adaptive immunity.

Histiocytes are white blood cells of the monocytic lineage and include macrophages and dendritic cells. In patients with a variety of infectious and noninfectious inflammatory disorders, histiocytes can engulf nonapoptotic leukocytes and nonsenescent erythrocytes and thus become hemophagocytes. We report here the identification and characterization of splenic hemophagocytes in a natural model of murine typhoid fever. The development of a flow-cytometric method allowed us to identify hemophagocytes based on their greater than 6N (termed 6N؉) DNA content. Characterization of the 6N؉ population from infected mice showed that these cells consist primarily of macrophages rather than dendritic cells and contain T lymphocytes, consistent with hemophagocytosis. Most 6N؉ macrophages from Salmonella enterica serovar Typhimurium-infected mice contain intact DNA, consistent with hemophagocytosis. In contrast, most 6N؉ macrophages from control mice or mice infected with a different bacterial pathogen, Yersinia pseudotuberculosis, contain damaged DNA. Finally, 6N؉ splenic macrophages from S. Typhimuriuminfected mice express markers consistent with an anti-inflammatory M2 activation state rather than a classical M1 activation state. We conclude that macrophages are the predominant splenic hemophagocyte in this disease model but not in Y. pseudotuberculosis-infected mice. The anti-inflammatory phenotype of hemophagocytic macrophages suggests that these cells contribute to the shift from acute to chronic infection.

e The Gram-negative intracellular bacterium Salmonella enterica serovar Typhimurium causes persistent systemic inflammatory disease in immunocompetent mice. Following oral inoculation with S. Typhimurium, mice develop a hematopathological syndrome akin to typhoid fever with splenomegaly, microcytic anemia, extramedullary erythropoiesis, and increased hemophagocytic macrophages in the bone marrow, liver, and spleen. Additionally, there is marked loss of iron from the spleen, an unanticipated result, given the iron sequestration reported in anemia of inflammatory disease. To establish why tissue iron does not accumulate, we evaluated multiple measures of pathology for 4 weeks following oral infection in mice. We demonstrate a quantitative decrease in splenic iron following infection despite increased numbers of splenic phagocytes. Infected mice have increased duodenal expression of the iron exporter ferroportin-1, consistent with increased uptake of dietary iron. Liver and splenic macrophages also express high levels of ferroportin-1. These observations indicate that splenic and hepatic macrophages export iron during S. Typhimurium infection, in contrast to macrophage iron sequestration observed in anemia of inflammatory disease. Tissue macrophage export of iron occurs concurrent with high serum concentrations of interferon gamma (IFN-␥) and interleukin 12 (IL-12). In individual mice, high concentrations of both proinflammatory (tumor necrosis factor alpha [TNF-␣])and anti-inflammatory (IL-10) cytokines in serum correlate with increased tissue bacterial loads throughout 4 weeks of infection. These in vivo observations are consistent with previous cell culture studies and suggest that the relocation of iron from tissue macrophages during infection may contribute to anemia and also to host survival of acute S. Typhimurium infection.T yphoid fever is acquired upon oral ingestion of food or water contaminated with Salmonella enterica serovar Typhi or Paratyphi and remains a serious threat to public health, especially in developing countries (1). Human typhoid fever causes a broad range of clinical signs, including splenomegaly, neuropathy, and hematopathology, such as cytopenias (2), that have been modeled in laboratory mice infected with Salmonella enterica serovar Typhimurium (3). Inbred mouse strains with differing genetic backgrounds are characterized as either sensitive or resistant to S. Typhimurium. Sensitivity to the intracellular bacterial infection is caused by genetic deficiencies in the innate or adaptive immune system (4, 5). For instance, mice lacking the cation transporter Nramp1 (Slc11a1), a regulator of cellular iron metabolism, are extremely sensitive to infection. Nramp1 ϩ mice are considered resistant to S. Typhimurium, because infection is generally nonfatal (6-8). Resistant mice infected by natural oral inoculation develop systemic infection with an acute hematopathological syndrome similar to that of humans with typhoid fever, including fever, anemia, inflammatory disease characterized by periph...