Raymond M. Johnson

Murine Oviduct Epithelial Cell Cytokine Responses to Chlamydia muridarum Infection Include Interleukin-12-p70 Secretion

ABSTRACT Epithelial cells play an important role in host defense as sentinels for invading microbial pathogens. Chlamydia trachomatis is an intracellular bacterial pathogen that replicates in reproductive tract epithelium. Epithelial cells lining the reproductive tract likely play a key role in triggering inflammation and adaptive immunity during Chlamydia infections. For this report a murine oviduct epithelial cell line was derived in order to determine how epithelial cells influence innate and adaptive immune responses during Chlamydia infections. As expected, oviduct epithelial cells infected by Chlamydia muridarum produced a broad spectrum of chemokines, including CXCL16, and regulators of the acute-phase response, including interleukin-1α (IL-1α), IL-6, and tumor necrosis factor alpha. In addition, infected epithelial cells expressed cytokines that augment gamma interferon (IFN) production, including IFN-α/β and IL-12-p70. To my knowledge this is the first report of a non-myeloid/lymphoid cell type making IL-12-p70 in response to an infection. Equally interesting, infected epithelial cells significantly upregulated transforming growth factor alpha precursor expression, suggesting a mechanism by which they might play a direct role in the pathological scarring seen as a consequence of Chlamydia infections. Data from these in vitro studies predict that infected oviduct epithelium contributes significantly to host innate and adaptive defenses but may also participate in the immunopathology seen with Chlamydia infections.

Pattern Recognition Molecules Activated by Chlamydia muridarum Infection of Cloned Murine Oviduct Epithelial Cell Lines

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the United States and a major cause of female infertility due to infection-induced Fallopian tube scarring. Epithelial cells are likely central to host defense and pathophysiology as they are the principal cell type productively infected by C. trachomatis. We generated cloned murine oviduct epithelial cell lines without viral or chemical transformation to investigate the role of the TLRs and cytosolic nucleotide binding site/leucine-rich repeat proteins Nod1 and Nod2 in epithelial responses to Chlamydia muridarum infection. RT-PCR assays detected mRNA for TLR2 (TLRs 1 and 6), TLR3, and TLR5. No mRNA was detected for TLRs 4, 7, 8, and 9. Messenger RNAs for Nod1 and Nod2 were present in the epithelial cell lines. Oviduct epithelial cell lines infected with C. muridarum or exposed to the TLR2 agonist peptidoglycan secreted representative acute phase cytokines IL-6 and GM-CSF in a MyD88-dependent fashion. Infected epithelial cell lines secreted the immunomodulatory cytokine IFN-β, even though C. muridarum does not have a clear pathogen-associated molecular pattern (PAMP) for triggering IFN-β transcription. The oviduct epithelial lines did not secrete IFN-β in response to the TLR2 agonist peptidoglycan or to the TLR3 agonist poly(I:C). Our data identify TLR2 as the principal TLR responsible for secretion of acute phase cytokines by C. muridarum-infected oviduct epithelial cell lines. The pattern recognition molecule responsible for infection-induced IFN-β secretion by oviduct epithelial cells remains to be determined.

Chlamydia muridarum Infection Elicits a Beta Interferon Response in Murine Oviduct Epithelial Cells Dependent on Interferon Regulatory Factor 3 and TRIF

ABSTRACT Chlamydia trachomatis is the most common sexually transmitted bacterial infection in the United States. Utilizing cloned murine oviduct epithelial cell lines, we previously identified Toll-like receptor 2 (TLR2) as the principal epithelial pattern recognition receptor (PRR) for infection-triggered release of the acute inflammatory cytokines interleukin-6 and granulocyte-macrophage colony-stimulating factor. The infected oviduct epithelial cell lines also secreted the immunomodulatory cytokine beta interferon (IFN-β) in a largely MyD88-independent manner. Although TLR3 was the only IFN-β production-capable TLR expressed by the oviduct cell lines, we were not able to determine whether TLR3 was responsible for IFN-β production because the epithelial cells were unresponsive to the TLR3 ligand poly(I-C), and small interfering RNA (siRNA) techniques were ineffective at knocking down TLR3 expression. To further investigate the potential role of TLR3 in the infected epithelial cell secretion of IFN-β, we examined the roles of its downstream signaling molecules TRIF and IFN regulatory factor 3 (IRF-3) using a dominant-negative TRIF molecule and siRNA specific for TRIF and IRF-3. Antagonism of either IRF-3 or TRIF signaling significantly decreased IFN-β production. These data implicate TLR3, or an unknown PRR utilizing TRIF, as the source of IFN-β production by Chlamydia-infected oviduct epithelial cells.

Improvement in HIV-related endothelial dysfunction using the anti-inflammatory agent salsalate: A pilot study

We hypothesized that heightened systemic inflammation contributes to the increased rate of cardiovascular events in HIV-infected patients not receiving combination antiretroviral therapy. We performed a pilot trial to assess the effects of the nuclear factor-κB inhibitor salsalate on flow-mediated dilation of the brachial artery, a measure of endothelial function. Flow-mediated dilation significantly improved after 8 weeks of salsalate. However, hepatotoxicity occurred frequently. Research using alternative agents is warranted to examine the role of inflammation in HIV-related cardiovascular disease.

Kawasaki-like syndromes and other vasculitic syndromes in HIV-infected patients.

Excluding drug-related hypersensitivity reactions, vasculitic syndromes are not common in HIV-positive patients. Review of the existing literature suggests that HIV positive patients may be predisposed to polyarteritis nodosa, microscopic polyarteritis, Kawasaki-like syndromes, acute occlusion syndromes, primary angiitis of the central nervous system and erythema elevatum diutinum. With the exception of erythema elevatum diutinum, these vasculitic syndromes have significant morbidities and mortality if they are not treated. It is therefore important to make these diagnoses and to initiate appropriate therapy in a timely fashion. Because fevers, malaise, weakness, rashes, headaches and neurologic symptoms are common in HIV-positive patients, it is probable that some cases of vasculitis go undiagnosed. In this report, we review vasculitic syndromes seen in HIV-positive patients. We also re-examine a previously published case of a young HIV-positive male who died of an acute myocardial infarction without atherosclerotic disease. Immunohistopathology of the affected arteries suggests that he died of a Kawasaki-like syndrome.

Chlamydia-specific CD4 T cell clones control Chlamydia muridarum replication in epithelial cells by nitric oxide-dependent and -independent mechanisms.

Chlamydia trachomatis serovars D–K are sexually transmitted intracellular bacterial pathogens that replicate in epithelial cells lining the human reproductive tract. It is clear from knockout mice and T cell depletion studies using Chlamydia muridarum that MHC class II and CD4 T cells are critical for clearing bacteria from the murine genital tract. It is not clear how CD4 T cells interact with infected epithelial cells to mediate bacterial clearance in vivo. Previous work using an epithelial tumor cell line showed that a Chlamydia-specific CD4 T cell clone was able to inhibit C. muridarum replication in vitro via induction of epithelial NO production. We have previously shown that Chlamydia-specific CD4 T cell clones can recognize and be activated by infected reproductive tract epithelial cells and block Chlamydia replication in them. We extend those observations by investigating the mechanism used by a panel of CD4 T cell clones to control Chlamydia replication in epithelial cells. We found that Chlamydia-specific CD4 T cell clones were cytolytic, but that cytolysis was not likely critical for controlling C. muridarum replication. For one, CD4 T cell clone-induced epithelial NO production was critical for controlling replication; however, the most potent CD4 T cell clones were dependent on T cell degranulation for replication control with only a minor additional contribution from NO production. We discuss our data as they relate to existing knockout mouse studies addressing mechanisms of T cell-mediated control of Chlamydia replication and their implications for intracellular epithelial pathogens in mouse models.

The Chlamydia muridarum-Induced IFN-β Response Is TLR3-Dependent in Murine Oviduct Epithelial Cells

Epithelial cells lining the murine genital tract act as sentinels for microbial infection, play a major role in the initiation of the early inflammatory response, and can secrete factors that modulate the adaptive immune response when infected with Chlamydia. C. muridarum-infected murine oviduct epithelial cells secrete the inflammatory cytokines IL-6 and GM-CSF in a TLR2-dependent manner. Further, C. muridarum infection induces IFN-β synthesis in the oviduct epithelial cells in a TRIF-dependent manner. Because murine oviduct epithelial cells express TLR3 but not TLRs 4, 7, 8, or 9, we hypothesized that TLR3 or an unknown TRIF-dependent pattern recognition receptor was the critical receptor for IFN-β production. To investigate the role of TLR3 in the Chlamydia-induced IFN-β response in oviduct epithelial cells, we used small interfering RNA, dominant-negative TLR3 mutants, and TLR3-deficient oviduct epithelial cells to show that the IFN-β secreted during C. muridarum infection requires a functional TLR3. Interestingly, we demonstrate that the TLR3 signaling pathway is not required for IFN-β synthesis in C. muridarum-infected macrophages, suggesting that there are alternate and redundant pathways to Chlamydia-induced IFN-β synthesis that seem to be dependent upon the cell type infected. Finally, because there is no obvious dsRNA molecule associated with Chlamydia infection, the requirement for TLR3 in Chlamydia-induced IFN-β synthesis in infected oviduct epithelial cells implicates a novel ligand that binds to and signals through TLR3.

Tissue-Resident T Cells as the Central Paradigm of Chlamydia Immunity.

ABSTRACT For almost 2 decades, results from Chlamydia pathogenesis investigations have been conceptualized using a cytokine polarization narrative. Recent viral immunity studies identifying protective tissue-resident memory T cells (Trm) suggest an alternative paradigm based on localized immune networks. As Chlamydia vaccines enter the preclinical pipeline and, in the case of an attenuated trachoma vaccine, are given to human subjects, it may be useful to ask whether cytokine polarization is the appropriate framework for understanding and evaluating vaccine efficacy. In this review, we revisit C. trachomatis pathogenesis data from mice and humans using a Trm narrative and note a comfortable concordance with the Chlamydia pathogenesis literature.

Successful treatment of CMV ventriculitis immune reconstitution syndrome

In HIV infected patients, cytomegalovirus (CMV) disease of the central nervous system is usually seen when the CD4+ count is less than 100 cells/mm3. Recommended treatment includes intravenous ganciclovir, foscarnet, or both.1 Ventriculitis is an uncommon but severe manifestation of CMV disease. Mortality is extremely high despite timely initiation of treatment. Little is known about CMV ventriculoencephalitis in the highly active antiretroviral therapy (HAART) era. A 38 year old woman with no significant past medical history was diagnosed with Pneumocystis jiroveci (Pneumocystis carinii) pneumonia as her AIDS defining illness in April 2002. On 15 July 2002, HAART was initiated with zidovudine, lamivudine, and efavirenz. Her CD4+ count was 42 cells/mm3 and her HIV-1 RNA level was 95 000 copies/ml. Serum CMV IgG was positive. Ten days later, she was evaluated for headache of new onset. Computed tomography (CT) of her head was unremarkable. Cerebrospinal fluid analysis showed no white cells, a red cell count of 10/mm3, a protein level of 58 mg/dl, and a glucose of 40 mg/ml. Cryptococcal antigen serology was negative. Two weeks later, after full compliance with her HIV therapy, she returned with headache, nausea, vomiting, and fever. Her temperature was 38.6°C (101.4°F). Neurological examination was normal and there was no meningism. Ophthalmological examination of the retinae with fully …

Chlamydia muridarum-Specific CD4 T-Cell Clones Recognize Infected Reproductive Tract Epithelial Cells in an Interferon-Dependent Fashion

ABSTRACT During natural infections Chlamydia trachomatis urogenital serovars replicate predominantly in the epithelial cells lining the reproductive tract. This tissue tropism poses a unique challenge to host cellar immunity and future vaccine development. In the experimental mouse model, CD4 T cells are necessary and sufficient to clear Chlamydia muridarum genital tract infections. This implies that resolution of genital tract infection depends on CD4 T-cell interactions with infected epithelial cells. However, no laboratory has shown that Chlamydia-specific CD4 T cells can recognize Chlamydia antigens presented by major histocompatibility complex class II (MHC-I) molecules on epithelial cells. In this report we show that MHC-II-restricted Chlamydia-specific CD4 T-cell clones recognize infected upper reproductive tract epithelial cells as early as 12 h postinfection. The timing of recognition and degree of T-cell activation are dependent on the interferon (IFN) milieu. Beta IFN (IFN-β) and IFN-γ have different effects on T-cell activation, with IFN-β blunting IFN-γ-induced upregulation of epithelial cell surface MHC-II and T-cell activation. Individual CD4 T-cell clones differed in their degrees of dependence on IFN-γ-regulated MHC-II for controlling Chlamydia replication in epithelial cells in vitro. We discuss our data as they relate to published studies with IFN knockout mice, proposing a straightforward interpretation of the existing literature based on CD4 T-cell interactions with the infected reproductive tract epithelium.