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Fulltext Lung-infiltrating T helper 17 cells as the major source of interleukin-17A production during pulmonary Cryptococcus neoformans infection

Movahed E, Cheok YY, Tan GMY, Lee CYQ, Cheong HC, Velayuthan RD, et al.

BMC Immunol, 2018 Nov 08;19(1):32.
PMID: 30409128 DOI: 10.1186/s12865-018-0269-5

BACKGROUND: IL-17A has emerged as a key player in the pathologies of inflammation, autoimmune disease, and immunity to microbes since its discovery two decades ago. In this study, we aim to elucidate the activity of IL-17A in the protection against Cryptococcus neoformans, an opportunistic fungus that causes fatal meningoencephalitis among AIDS patients. For this purpose, we examined if C. neoformans infection triggers IL-17A secretion in vivo using wildtype C57BL/6 mice. In addition, an enhanced green fluorescence protein (EGFP) reporter and a knockout (KO) mouse models were used to track the source of IL-17A secretion and explore the protective function of IL-17A, respectively.
RESULTS: Our findings showed that in vivo model of C. neoformans infection demonstrated induction of abundant IL-17A secretion. By examining the lung bronchoalveolar lavage fluid (BALF), mediastinal lymph node (mLN) and spleen of the IL-17A-EGFP reporter mice, we showed that intranasal inoculation with C. neoformans promoted leukocytes lung infiltration. A large proportion (~ 50%) of the infiltrated CD4+ helper T cell population secreted EGFP, indicating vigorous TH17 activity in the C. neoformans-infected lung. The infection study in IL-17A-KO mice, on the other hand, revealed that absence of IL-17A marginally boosted fungal burden in the lung and accelerated the mouse death.
CONCLUSION: Therefore, our data suggest that IL-17A is released predominantly from TH17 cells in vivo, which plays a supporting role in the protective immunity against C. neoformans infection.
Fulltext Podoplanin Drives Motility of Active Macrophage via Regulating Filamin C During Helicobacter pylori Infection

Cheok YY, Tan GMY, Fernandez KC, Chan YT, Lee CYQ, Cheong HC, et al.

Front Immunol, 2021;12:702156.
PMID: 34707599 DOI: 10.3389/fimmu.2021.702156

Podoplanin (Pdpn) is a mucin-type transmembrane protein that has been implicated in multiple physiological settings including lymphangiogenesis, platelet aggregation, and cancer metastasis. Here, we reported an absence of Pdpn transcript expression in the resting mouse monocytic macrophages, RAW264.7 cells; intriguingly, a substantial upregulation of Pdpn was observed in activated macrophages following Helicobacter pylori or lipopolysaccharide stimulation. Pdpn-knockout macrophages demonstrated intact phagocytic and intracellular bactericidal activities comparable to wild type but exhibited impaired migration due to attenuated filopodia formation. In contrast, an ectopic expression of Pdpn augmented filopodia protrusion in activated macrophages. NanoString analysis uncovered a close dependency of Filamin C gene on the presence of Pdpn, highlighting an involvement of Filamin C in modulation of actin polymerization activity, which controls cell filopodia formation and migration. In addition, interleukin-1β production was significantly declined in the absence of Pdpn, suggesting a role of Pdpn in orchestrating inflammation during H. pylori infection besides cellular migration. Together, our findings unravel the Pdpn network that modulates movement of active macrophages.
Influx of podoplanin-expressing inflammatory macrophages into the genital tract following Chlamydia infection

Chan YT, Cheok YY, Cheong HC, Tan GMY, Seow SR, Tang TF, et al.

Immunol Cell Biol, 2023 Apr;101(4):305-320.
PMID: 36658328 DOI: 10.1111/imcb.12621

Genital Chlamydia trachomatis infection remains a major health issue as it causes severe complications including pelvic inflammatory disease, ectopic pregnancy and infertility in females as a result of infection-associated chronic inflammation. Podoplanin, a transmembrane receptor, has been previously reported on inflammatory macrophages. Thus, strategies that specifically target podoplanin might be able to reduce local inflammation. This study investigated the expression level and function of podoplanin in a C. trachomatis infection model. C57BL/6 mice infected with the mouse pathogen Chlamydia muridarum were examined intermittently from days 1 to 60 using flow cytometry analysis. Percentages of conventional macrophages (CD11b+ CD11c- F4/80+ ) versus inflammatory macrophages (CD11b+ CD11c+ F4/80+ ), and the expression of podoplanin in these cells were investigated. Subsequently, a podoplanin-knockout RAW264.7 cell was used to evaluate the function of podoplanin in C. trachomatis infection. Our findings demonstrated an increased CD11b+ cell volume in the spleen at day 9 after the infection, with augmented podoplanin expression, especially among the inflammatory macrophages. A large number of podoplanin-expressing macrophages were detected in the genital tract of C. muridarum-infected mice. Furthermore, analysis of the C. trachomatis-infected patients demonstrated a higher percentage of podoplanin-expressing monocytes than that in the noninfected controls. Using an in vitro infection in a transwell migration assay, we identified that macrophages deficient in podoplanin displayed defective migratory function toward C. trachomatis-infected HeLa 229 cells. Lastly, using immunoprecipitation-mass spectrometry method, we identified two potential podoplanin interacting proteins, namely, Cofilin 1 and Talin 1 actin-binding proteins. The present study reports a role of podoplanin in directing macrophage migration to the chlamydial infection site. Our results suggest a potential for reducing inflammation in individuals with chronic chlamydial infections by targeting podoplanin.
CPAF, HSP60 and MOMP antigens elicit pro-inflammatory cytokines production in the peripheral blood mononuclear cells from genital Chlamydia trachomatis-infected patients

Cheong HC, Lee CYQ, Cheok YY, Shankar EM, Sabet NS, Tan GMY, et al.

Immunobiology, 2019 01;224(1):34-41.
PMID: 30477893 DOI: 10.1016/j.imbio.2018.10.010

BACKGROUND: Persistent inflammation caused by Chlamydia trachomatis in the female genital compartment represents one of the major causes of pelvic inflammatory disease (PID), ectopic pregnancy and infertility in females. Here, we examined the pro-inflammatory cytokine response following stimulation with three different types of C. trachomatis antigens, viz. chlamydial protease-like factor (CPAF), heat shock protein 60 (HSP60) and major outer membrane protein (MOMP).
METHODS: A total of 19 patients with genital C. trachomatis infection and 10 age-matched healthy controls were recruited for the study. Peripheral blood mononuclear cells (PBMCs) isolated from genital C. trachomatis-infected females were cultured in the presence of CPAF, HSP60 and MOMP antigens, and cytokines were measured by ELISA assay.
RESULTS: We reported that pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were robustly secreted following antigenic exposure. Notably, CPAP and MOMP were more potent in triggering IL-1β, as compared to HSP60. Elevated levels of the proinflammatory cytokines were also noted in the samples infected with plasmid-bearing C. trachomatis as compared to those infected with plasmid-free strains.
CONCLUSIONS: Our study highlights distinct ability of chlamydial antigens in triggering pro-inflammatory response in the host immune cells.
Fulltext Diversity of endocervical microbiota associated with genital Chlamydia trachomatis infection and infertility among women visiting obstetrics and gynecology clinics in Malaysia

Cheong HC, Yap PSX, Chong CW, Cheok YY, Lee CYQ, Tan GMY, et al.

PLoS One, 2019;14(11):e0224658.
PMID: 31738795 DOI: 10.1371/journal.pone.0224658

The cervical microbiota constitutes an important protective barrier against the invasion of pathogenic microorganisms. A disruption of microbiota within the cervical milieu has been suggested to be a driving factor of sexually transmitted infections. These include Chlamydia trachomatis which frequently causes serious reproductive sequelae such as infertility in women. In this study, we profiled the cervical microbial composition of a population of 70 reproductive-age Malaysian women; among which 40 (57.1%) were diagnosed with genital C. trachomatis infection, and 30 (42.8%) without C. trachomatis infection. Our findings showed a distinct compositional difference between the cervical microbiota of C. trachomatis-infected subjects and subjects without C. trachomatis infection. Specifically, significant elevations of mostly strict and facultative anaerobes such as Streptococcus, Megasphaera, Prevotella, and Veillonella in the cervical microbiota of C. trachomatis-positive women were detected. The results from the current study highlights an interaction of C. trachomatis with the environmental microbiome in the endocervical region.