Induction of Interferon-Stimulated Genes by Chlamydia pneumoniae in Fibroblasts Is Mediated by Intracellular Nucleotide-Sensing Receptors

BACKGROUND: Recognition of microorganisms by the innate immune system is mediated by pattern recognition receptors, including Toll-like receptors and cytoplasmic RIG-I-like receptors. Chlamydia, which include several human pathogenic species, are obligate intracellular gram-negative bacteria that replicate in cytoplasmic vacuoles. The infection triggers a host response contributing to both bacterial clearance and tissue damage. For instance, type I interferons (IFN)s have been demonstrated to exacerbate the course of Chlamydial lung infections in mice. METHODS/PRINCIPAL FINDINGS: Here we show that Chlamydia pneumoniae induces expression of IFN-stimulated genes (ISG)s dependent on recognition by nucleotide-sensing Toll-like receptors and RIG-I-like receptors, localized in endosomes and the cytoplasm, respectively. The ISG response was induced with a delayed kinetics, compared to virus infections, and was dependent on bacterial replication and the bacterial type III secretion system (T3SS). CONCLUSIONS/SIGNIFICANCE: Activation of the IFN response during C. pneumoniae infection is mediated by intracellular nucleotide-sensing PRRs, which operate through a mechanism dependent on the bacterial T3SS. Strategies to inhibit the chlamydial T3SS may be used to limit the detrimental effects of the type I IFN system in the host response to Chlamydia infection

Induction of ISGs by <i>Chlamydia pneumoniae</i> is dependent on TBK1/IKKε, bacterial replication and the T3SS.

<p>(A, C) WT, tbk1^−/− ikke^−/− and atg5^−/− MEFs were infected with <i>C. pneumoniae</i>, MOI 5, for 36 h before harvest of total RNA. (B) WT MEFs were infected with live or UV-inactivated <i>C. pneumoniae</i> (CFU/cell 5) in the presence and absence of ampicillin (20 µg/ml) as indicated. (D) The cells were treated with INP0341 (60 µM), INP0406 (60 µM) or DMSO (vehicle) and infected with <i>C. pneumoniae</i>, MOI 5. Total RNA was harvested 36 hrs post-infection. (A–D) Levels of ISG56 mRNA were determined by real-time PCR. The measured ISG56 mRNA levels were normalized to β-actin, and data are presented as means of triplicate cultures +/− st.dev. Similar results were obtained in 2–3 independent experiments. RU, relative units.</p

<i>Chlamydia pneumoniae</i>-induced expression of ISGs is dependent on bacterial recognition by endosomal and cytoplasmic nucleotide-sensing PRRs.

<p>(A–F) WT, myd88^−/− trif^−/− and mavs^−/− MEFs were infected with (A–B) <i>C. pneumoniae</i>, MOI 5, for 36 h (C–D) HSV-2, MOI 1, 6 hrs, or (E–F) SeV, MOI 1, 6 hrs. Total RNA was harvested and levels of ISG56 mRNA were determined by real-time PCR. (G) MEFs were stimulated with polyIC (25 µg/ml), LPS (200 ng/ml), and ODN1826 (1 µM) alone or in combination as indicated. Total RNA was harvested 6 hrs post-stimulation, and levels of ISG56 mRNA were determined by real-time PCR. (H) The cells were infected with <i>C. pneumonia</i>, MOI 5, for 18 h in the presence or absence of polymyxin B (50 µg/ml) or chloroquine (10 µM) before harvest of total mRNA. The measured ISG56 mRNA levels were normalized to β-actin, and data are presented as means of triplicate cultures +/− st.dev. Similar results were obtained in 2–3 independent experiments. RU, relative units.</p

<i>Chlamydia pneumoniae</i> induces expression of ISGs with a delayed kinetics compared to viruses.

<p>(A) MEF cells were infected with <i>C. pneumoniae</i> (5 UFU/cell) in presence or absence of cycloheximide (CHX, 1 µg/ml). 36 hrs post infection, the cells were fixed and stained with an antibody against chlamydial LPS and fluorescence/Nomaski images were obtained. (B–E) MEFs were infected with (B–C) <i>C. pneumonia</i>, MOI 5(D) HSV-2, MOI 1, or (E) SeV, MOI 1 for the indicated time intervals. Total RNA was harvested and mRNA levels of ISG56 and CCL5 were determined by real-time PCR and normalized to β-actin. The data are presented as means of triplicate cultures +/− st.dev. Similar results were obtained in 2–5 independent experiments. RU, relative units. Infection-induced gene expression significantly above the expression induced by mock infection at the same tine point (p<0.05) is marked with an asterisk, *.</p

Targeting IL-23 for the interception of obesity-associated colorectal cancer

Inflammation and obesity are two major factors that promote Colorectal cancer (CRC). Our recent data suggests that interleukin (IL)-23, is significantly elevated in CRC tumors and correlates with patient obesity, tumor grade and survival. Thus, we hypothesize that obesity and CRC may be linked via inflammation and IL-23 may be a potential target for intervention in high-risk patients. TCGA dataset and patient sera were evaluated for IL-23A levels. IL-23A [IL-23 p19−/−] knockout (KO) mice were crossed to Apcmin/+ mice and progeny were fed low-fat or high-fat diets. At termination intestines were evaluated for tumorigenesis. Tumors, serum, and fecal contents were analyzed for protein biomarkers, cytokines, and microbiome profile respectively. IL-23A levels are elevated in the sera of patients with obesity and colon tumors. Genetic ablation of IL-23A significantly suppressed colonic tumor multiplicity (76–96 %) and incidence (72–95 %) in male and female mice. Similarly, small-intestinal tumor multiplicity and size were also significantly reduced in IL-23A KO mice. IL-23A knockdown in Apcmin/+ mice fed high-fat diet, also resulted in significant suppression of colonic (50–58 %) and SI (41–48 %) tumor multiplicity. Cytokine profiling showed reduction in several circulating pro-inflammatory cytokines including loss of IL-23A. Biomarker analysis suggested reduced tumor cell proliferation and immune modulation with an increase in tumor-infiltrating CD4+ and CD8+ T-lymphocytes in the IL-23A KO mice compared to controls. Fecal microbiome analysis revealed potentially beneficial changes in the bacterial population profile. In summary, our data indicates a tumor promoting role for IL-23 in CRC including diet-induced obesity. With several IL-23 targeted therapies in clinical trials, there is a great potential for targeting this cytokine for CRC prevention and therapy

RIG-I-mediated activation of p38 MAPK is essential for viral induction of interferon and activation of dendritic cells:dependence on TRAF2 and TAK1

The innate immune system provides an initial defense system against microbial infections and contributes to the development of adaptive immune response. Type I interferons play a pivotal role for the first line of defense against virus infections, and dendritic cells (DCs) are important sensors of pathogens responsible for priming of adaptive immune responses in lymphoid organs. Here we have investigated the role and mechanisms of activation of the MAPK pathway in innate immune responses induced by Sendai virus, a negative sense single-stranded RNA virus. Both p38 and JNK were activated in fibroblasts and DCs after infection with Sendai virus in a manner dependent on virus replication and RIG-I. Virus replication was also required for stimulation of interferon production in both cell types and interleukin-12 production in DCs. Blocking of p38 MAPK activation by the specific inhibitor SB202190 abolished the expression of these cytokines. p38 MAPK exerted its function independent of the MAPK-activated protein kinases MK2, MNK, and MSK1/2. We also observed that TRAF2 and TAK1 were essential for RIG-I-mediated activation of p38 MAPK. Interestingly, the kinase activity of p38 MAPK was required for its own phosphorylation, which was kinetically associated with TAB1 interaction. By contrast, the canonical p38 upstream kinase MKK3 was not involved in the p38-dependent response. Thus, activation of p38 MAPK by RIG-I proceeds via a TRAF2-TAK1-dependent pathway, where the enzymatic activity of the kinase plays an essential role. The p38 MAPK in turn stimulates important processes in the innate antiviral response

Molecular disparities in colorectal cancers of White Americans, Alabama African Americans, and Oklahoma American Indians

Abstract In the US, the majority of cancer samples analyzed are from white people, leading to biases in racial and ethnic treatment outcomes. Colorectal cancer (CRC) incidence and mortality rates are high in Alabama African Americans (AAs) and Oklahoma American Indians (AIs). We hypothesized that differences between racial groups may partially explain these disparities. Thus, we compared transcriptomic profiles of CRCs of Alabama AAs, Oklahoma AIs, and white people from both states. Compared to CRCs of white people, CRCs of AAs showed (a) higher expression of cytokines and vesicle trafficking toward modulated antitumor-immune activity, and (b) lower expression of the ID1/BMP/SMAD axis, IL22RA1, APOBEC3, and Mucins; and AIs had (c) higher expression of PTGS2/COX2 (an NSAID target/pro-oncogenic inflammation) and splicing regulators, and (d) lower tumor suppressor activities (e.g., TOB2, PCGF2, BAP1). Therefore, targeting strategies designed for white CRC patients may be less effective for AAs/AIs. These findings illustrate needs to develop optimized interventions to overcome racial CRC disparities