Publications

We recently demonstrated that a new PG1828-encoded lipoprotein (PG1828LP) was able to be separated from a Porphyromonas gingivalis lipopolysaccharide (LPS) preparation, and we found that it exhibited strong cell activation, similar to that of Escherichia coli LPS, through a Toll-like receptor 2 (TLR2)-dependent pathway. In order to determine the virulence of PG1828LP toward cell activation, we generated a PG1828-deficient mutant of P. gingivalis strain 381 by allelic exchange mutagenesis using an ermF-ermAM antibiotic resistance cassette. A highly purified preparation of LPS from a PG1828-deficient mutant (DeltaPG1828-LPS) showed nearly the same ladder-like patterns in silver-stained gels as a preparation of LPS from a wild-type strain (WT-LPS), as well as Limulus amoebocyte lysate activities that were similar to those of the WT-LPS preparation. However, the ability of the DeltaPG1828-LPS preparation to activate NF-kappaB in TLR2-expressing cells was markedly attenuated. Cytokine production by human gingival fibroblasts was also decreased in response to the DeltaPG1828-LPS preparation in comparison with the WT-LPS preparation, and the activity was comparable to the stimulation of highly purified lipid A of P. gingivalis by TLR4. Further, lethal toxicity was rarely observed following intraperitoneal injection of the PG1828-deficient mutant into mice compared to that with the wild-type strain, while the DeltaPG1828-LPS preparation showed no lethal toxicity. Taken together, these results clearly indicate that PG1828LP plays an essential role in inflammatory responses and may be a major virulence factor of P. gingivalis.

Toll-like receptor (TLR)-mediated recognition of pathogens represents one of the most important mechanisms of innate immunity. A proximal signaling event of TLR is the direct binding of an adaptor protein MyD88 to TLR and recruitment of the IL-1R-associated kinase (IRAK). In the present study, we examined the effect of several TLR ligands on protein tyrosine phosphorylation in rat macrophages. Macrophage-activating lipopeptide-2 kDa (MALP2) and lipoarabinomannan were used as activators of TLR2, while lipopolysaccharides (LPS) and lipoteichoic acid were used as TLR4 ligands. All these ligands induced tyrosine phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) and its substrate paxillin, an integrin-associated focal adhesion adaptor protein, in the macrophages. PP2, an inhibitor of Src family tyrosine kinases, prevented the TLR-induced phosphorylation of paxillin and Pyk2 without affecting TLR-induced IRAK activation. MALP2 failed to induce paxillin phosphorylation in the macrophages from MyD88-knockout mice. In contrast, the effect of LPS weakened, but was still observed even in the MyD88-deficient cells. Thus, TLR regulate the function of paxillin in an Src family-dependent mechanism through both MyD88-dependent and MyD88-independent pathways.

Volume 13, no. 2, e03197-21, 2022, https://doi.org/10.1128/mbio.03197-21. An investigation conducted by the corresponding author's laboratory has concluded that the data shown in Fig. 3D–F, 4A–E and G, 5, S1, S2, S4, and S5 are the results of fabricated experiments or extensive falsification. An official investigation is ongoing at Osaka University, and certain results will require further verification by re-experimentation. Therefore, we are retracting the article and regret any inconvenience this may have caused.

Effective activation of dendritic cells (DCs) toward T helper (Th)-1 cell polarization would improve DC-based antitumor immunotherapy, helping promote the development of immunotherapeutic vaccines based on T-cell immunity. To achieve this goal, it is essential to develop effective immune adjuvants that can induce powerful Th1 cell immune responses. The pathogenic organism Mycobacterium tuberculosis includes certain constitutes, such as heparin-binding hemagglutinin (HBHA), that possess a strong immunostimulatory potential. In this study, we report the first clarification of the functions and precise mechanism of HBHA in immune stimulation settings relevant to cancer. HBHA induced DC maturation in a TLR4-dependent manner, elevating expression of the surface molecules CD40, CD80, and CD86, MHC classes I and II and the proinflammatory cytokines IL-6, IL-12, IL-1β, TNF-α, and CCR7, as well as stimulating the migratory capacity of DCs in vitro and in vivo. Mechanistic investigations established that MyD88 and TRIF signaling pathways downstream of TLR4 mediated secretion of HBHA-induced proinflammatory cytokines. HBHA-treated DCs activated naïve T cells, polarized CD4(+) and CD8(+) T cells to secrete IFN-γ, and induced T-cell-mediated cytotoxicity. Notably, systemic administration of DCs that were HBHA-treated and OVA(251-264)-pulsed ex vivo greatly strengthened immune priming in vivo, inducing a dramatic regression of tumor growth associated with long-term survival in a murine E.G7 thymoma model. Together, our findings highlight HBHA as an immune adjuvant that favors Th1 polarization and DC function for potential applications in DC-based antitumor immunotherapy.

Host resistance to the intracellular protozoan parasite Trypanosoma cruzi depends on IFN-γ production by T cells and NK cells. However, the involvement of innate immunity in host resistance to T. cruzi remains unclear. In the present study, we investigated host defense against T. cruzi by focusing on innate immunity. Macrophages and dendritic cells (DCs) from MyD88−/−TRIF−/− mice, in which TLR-dependent activation of innate immunity was abolished, were defective in the clearance of T. cruzi and showed impaired induction of IFN-β during T. cruzi infection. Neutralization of IFN-β in MyD88−/− macrophages led to enhanced T. cruzi growth. Cells from MyD88−/−IFNAR1−/− mice also showed impaired T. cruzi clearance. Furthermore, both MyD88−/−TRIF−/− and MyD88−/−IFNAR1−/− mice were highly susceptible to in vivo T. cruzi infection, highlighting the involvement of innate immune responses in T. cruzi infection. We further analyzed the molecular mechanisms for the IFN-β-mediated antitrypanosomal innate immune responses. MyD88−/−TRIF−/− and MyD88−/−IFNAR1−/− macrophages and DCs exhibited defective induction of the GTPase IFN-inducible p47 (IRG47) after T. cruzi infection. RNA interference-mediated reduction of IRG47 expression in MyD88−/− macrophages resulted in increased intracellular growth of T. cruzi. These findings suggest that TLR-dependent expression of IFN-β is involved in resistance to T. cruzi infection through the induction of IRG47.

No abstract is provided for this article.

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b+ F4/80+ MHC-II+ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS+ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.

To determine the role of endogenous interleukin-18 (IL-18) in pneumococcal meningitis, meningitis was induced in IL-18 gene-deficient (IL-18−/−) and wild-type (WT) mice by intranasal inoculation of Streptococcus pneumoniae with hyaluronidase. Induction of meningitis resulted in an upregulation of both pro- and mature IL-18 in brain tissue in WT mice. IL-18−/− and WT mice were equally susceptible to develop meningitis after intranasal infection, yet IL-18−/− mice showed a prolonged survival and a suppressed inflammatory response, as reflected by a less profound inflammatory infiltrate around the meninges and lower concentrations of cytokines and chemokines in brain tissue. These findings suggest that endogenous IL-18 contributes to a detrimental inflammatory response during pneumococcal meningitis and that elimination of IL-18 may improve the outcome of this disease.

Chronic pancreatitis is a well-known risk factor for pancreatic ductal adenocarcinoma (PDA) development in humans, and inflammation promotes PDA initiation and progression in mouse models of the disease. However, the mechanistic link between inflammatory damage and PDA initiation is unclear. Using a Kras-driven mouse model of PDA, we establish that the inflammatory mediator Stat3 is a critical component of spontaneous and pancreatitis-accelerated PDA precursor formation and supports cell proliferation, metaplasia-associated inflammation, and MMP7 expression during neoplastic development. Furthermore, we show that Stat3 signaling enforces MMP7 expression in PDA cells and that MMP7 deletion limits tumor size and metastasis in mice. Finally, we demonstrate that serum MMP7 level in human patients with PDA correlated with metastatic disease and survival.

Tail DNA genotyping of fetal and neonatal mice from C/EBPβ heterozygous parents was performed to determine whether the decreased number of surviving C/EBPβ mutants was caused by prenatal or postnatal death. Eighty-four 3-week-old mice born of heterozygous parents had significantly lower numbers of C/EBPβ-deficient offspring than the expected Mendelian ratio (29.8%+/+, 65.5%+/-, 4.76%-/-, P<0.05). The genotypes of 72 fetal mice from intercrossed heterozygotes showed approximately the expected 1:2:1 Mendelian ratio (18.1% +/+, 52.8% +/-, 29.2% -/-, P>0.05). No difference in the proportions by sex could be detected in these perinates. This data indicates that C/EBPβ-deficient mice have unknown lethal problems between the embryonic stage and weaning.

No abstract is provided for this article.

We have reported that mPGES-1/PGE2 induced blood flow recovery from ischemia by promoting accumulation of Tregs. Expression of chemokines and the SDF-1/CXCR4 axis has been implicated in Tregs migration in angiogenesis. Therefore, we examined whether the SDF-1/CXCR4 axis plays a role in the accumulation of Tregs in angiogenesis.