New insights into Chlamydia pathogenesis: Role of leukemia inhibitory factor.

In addition to its physiological roles, the LIF/LIFR axis has been broadly discussed in the context of chronic inflammation, including chronic airway inflammation ( Knight, 2001 ), cutaneous inflammation ( Zhu et al., 2001 ), neuroinflammation ( Linker et al., 2008 ; Pan et al., 2008 ), and cancer-associated inflammation ( Christianson et al., 2021 ), mostly as an anti-inflammatory cytokine. In infection models, LIF is recognized as a vital stem cell growth factor that protects the lung from collateral damage during inflammatory attack against viral infections ( Quinton et al., 2012 ; Foronjy et al., 2014 ). The production of LIF during chlamydial infection has been reported by several in vitro and in vivo studies ( Hess et al., 2001 ; Peters et al., 2005 ; Refaat et al., 2016 ; Hou et al., 2018 ; Kessler et al., 2019 ). It was first identified through comparing the transcriptomic profile of 1176 genes in DNA arrays of Ct -infected and mock-infected epithelial HeLa cells, in which 18 genes, including LIF, were up-regulated by Ct infection ( Hess et al., 2001 ). This observation has been recently confirmed in human fallopian tube organoid cultures ( Kessler et al., 2019 ), which provide a novel in vitro system for recapitulating Ct infection in vivo . It was demonstrated that LIF is readily induced by Ct infection, along with robust activation of type I interferon (IFN-β) signaling and upregulation of inducible nitric oxide synthase (NOS2) to control Ct replication in organoids. In addition, LIF/LIFR signaling is involved in tissue injury and repair responses that are needed for maintaining epithelial homeostasis and organoid renewal ( Kessler et al., 2019 ). Therefore, LIF seems to be part of cell-autonomous immunity, and its production is a protective response. Notably, LIF production is only triggered in mice by a pathological, plasmid-containing Chlamydia muridarum , but not a plasmid-free one. The level of LIF is closely associated with the degree of bacterial ascension in the upper genital tract and the formation of hydrosalpinx ( Hou et al., 2018 ), a common tissue pathology associated with Ct infection in both humans and mice that can cause infertility. Similarly, LIF mRNA and protein are most detected at high levels in human fallopian tube samples obtained from ectopic pregnancies associated with Ct infections but not non- Ct infections ( Refaat et al., 2016 ). While these experimental results support a potential role of LIF in Ct pathogenesis, it is intriguing why a protective response is highly associated with tissue pathology. The human fallopian tube is a conduit that has a major functional role in oocyte pickup, fertilization, and embryo transport. The fallopian tube mucosa contains two major histologic cell types: ciliated epithelial cells and secretory epithelial cells, which work together for effective tubal transport of ova, sperm, and embryos for successful spontaneous pregnancy ( Lyons et al., 2006 ). Propulsion of gametes and embryos is achieved by complex interactions between muscle contractions, ciliary activity, and the flow of tubal secretions. Proper density of ciliated cells is required to avoid ectopic pregnancy ( Lyons et al., 2006 ). It has been demonstrated that fallopian tubes containing an ectopic pregnancy have a marked reduction in the number of ciliated cells in comparison with those of women with an intrauterine gestation ( Vasquez et al., 1983 ; Lyons et al., 2006 ). Detailed ultrastructural analysis of hydrosalpinx of infertile women also demonstrates severe abnormalities in epithelial cells, including flattening of the epithelial layer and severe loss of ciliated cells ( Ajonuma et al., 2005 ). Notably, ciliated cells and secretory cells are developmentally connected. A study using in vivo genetic cell lineage tracing in mice demonstrated that secretory epithelial cells not only self-renew, but also give rise to ciliated epithelial cells ( Ghosh et al., 2017 ). LIF/LIFR signaling has been shown to alter the fate of epithelial cells during Ct infection ( Kessler et al., 2019 ). The density of ciliated cells in organoids chronically infected with Ct is markedly reduced compared to non-infected controls ( Kessler et al., 2019 ). This is likely mediated by LIF, as addition of recombinant LIF protein to non-infected organoids potently inhibits the frequency of ciliated cells ( Kessler et al., 2019 ). The results from the organoid cultures provide strong experimental evidence supporting a potential role of LIF in epithelial transformation during Ct infection. LIF is also recognized as a cell-autonomous molecule during intracellular viral infections caused by HIV ( Patterson et al., 2002 ; Tjernlund et al., 2006 ) and HPV ( Bay et al., 2011 ). We were unable to find any reports on LIF production caused by extracellular pathogens, such as Neisseria gonorrhoeae . Therefore, it is likely that LIF is produced preferentially, if not exclusively, upon intracellular infections for combating intracellular pathogens and assisting in tissue repair and epithelium homeostasis. While this is a protective response in nature, repeated Ct infections and/or persistent Ct infections may lead to aberrant expression of LIF and LIF-mediated alterations of the epithelium, which has been demonstrated in in vitro models of persistent chlamydial infections ( Peters et al., 2005 ). Clinical observations indicate that most women with TFI and serological evidence of Ct infection lack a history of clinical PID ( Brunham et al., 1985 ). Therefore, it is likely that the regulation of LIF and LIF-mediated responses are operating for long periods of time, during which, LIF levels are constantly being modified by surrounding proinflammatory cytokines and growth factors ( Table 3 ). LIF expression is robustly induced by proinflammatory signals (e.g., LPS, IL-1 and TNF-α) ( Wetzler et al., 1991 ; Ishimi et al., 1992 ; Hamilton et al., 1993 ; Wetzler et al., 1994 ; Arici et al., 1995 ; Perrier d'Hauterive et al., 2004 ) and signaling molecules active in tissue growth and development, including platelet-derived growth factor (PDGF) ( Arici et al., 1995 ), epidermal growth factor (EGF) ( Arici et al., 1995 ), human chorionic gonadotropin (hCG) ( Perrier d'Hauterive et al., 2004 ), insulin-like growth factor (IGF) ( Perrier d'Hauterive et al., 2004 ), and transforming growth factor-β (TGF-β) ( Wetzler et al., 1991 ; Arici et al., 1995 ; Perrier d'Hauterive et al., 2004 ; Ruan et al., 2010 ; Ota et al., 2013 ). Many of these molecules are concurrently induced by Ct infection, which collectively amplify LIF expression and LIFR-mediated responses, and eventually lead to a marked reduction in ciliated epithelial cells and an increase of secretory epithelial cells in the fallopian tube. In comparison, IFN-γ potently supresses LIF expression in endometrial epithelial cells and stromal cells in vitro ( Arici et al., 1995 ). Of note, IL-4 is a typical type 2 cytokine that regulates LIF in a cell type-dependent manner. IL-4 downregulates LIF in cultured bone marrow stromal cells, synovial fibroblasts and liver myofibroblasts ( Wetzler et al., 1994 ; Denizot et al., 1999 ), whereas it upregulates LIF secretion in type 2 helper T-cells ( Piccinni et al., 1998 ). Given that IFN-γ and IL-4 are primarily produced by T cells, it is possible that LIF production may diminish upon the establishment of adaptive immune responses. While LIF has been shown to promote regulatory T cells and inhibit the differentiation of type 17 helper T-cells ( Metcalfe, 2011 ), it is unclear how T cell responses are regulated by prolonged LIF production during Ct infection. Although only limited knowledge is available, the crosstalk between LIF and T cells is likely an integral part of Ct pathogenesis and additional studies are warranted. Regulation of LIF expression. Based on the collective evidence discussed above, it is appealing to suggest that the LIF-mediated reduction in ciliated epithelial cell density is a key process of Ct pathogenesis, which can lead to reduced opportunities for fertilization and increased risk of ectopic pregnancy. Although it remains to be demonstrated experimentally, increased secretory cell density may lead to over-production and accumulation of fluids inside the fallopian tube, a characteristic feature of hydrosalpinx ( Ng and Cheong, 2019 ). Consistent with this notion, LIF is found to be expressed in human fallopian tubes ( Keltz et al., 1996 ) and is markedly elevated in chronically inflamed fallopian tubes ( Ji et al., 2009 ). Furthermore, ectopic pregnancies are mostly associated with intracellular infections caused by Ct and Mycoplasma genitalium , and less frequently linked to extracellular pathogens like Neisseria gonorrhoeae ( Ashshi et al., 2015 ; Refaat et al., 2016 ), despite its ability to cause a similar spectrum of pelvic inflammatory diseases ( Xu and Gray-Owen, 2021 ). The Human Protein Atlas dataset shows that high expression of LIF is a poor prognostic marker for human cervical cancer ( Human Protein Atlas, 2022 ), for which HPV and Ct are well-known risk factors ( Zhu et al., 2016 ; Liao et al., 2022 ). Ct infection is also a risk factor for ovarian cancer ( Hosseininasab-Nodoushan et al., 2021 ). While HPV promotes cervical cancer via oncogenic transformation ( Baedyananda et al., 2022 ), Ct may promote cervical cancer and ovarian cancer via aberrant LIF signaling, which has been shown to regulate multiple hallmarks of cancer, including proliferation, metastasis and chemoresistance ( Jones and Jenkins, 2018 ; Jorgensen and de la Puente, 2022 ). LIF also has a significant role in enriching and maintaining cancer stem cells, epithelial to mesenchymal transition, de-differentiation, and re-differentiation of cancer cells ( Halder et al., 2022 ).

The Chlamydiae family is a diverse group of obligatory intracellular bacteria, comprised of both pathogens and commensals, and found in varying habitats that can extend as far as the bottom of the Arctic Ocean ( Dharamshi et al., 2020 ). Chlamydia trachomatis ( Ct ) is a human pathogen and the most common bacterial cause of sexually transmitted infections worldwide ( WHO, 2021 ). An uptick in the reporting of chlamydial urogenital infections began in the 1990s following improvements in diagnostic technologies, and the rates have been increasing annually ever since ( CDC, 2020 ; Huai et al., 2020 ; Control ECfDPa, 2022 ). Ct affects mostly young women aged 15-24, but can infect both men and women of all age groups ( CDC, 2020 ; Huai et al., 2020 ; Control ECfDPa, 2022 ). Urogenital Ct infection is clinically associated with cervicitis, urethritis, endometritis, and salpingitis in women, and urethritis, proctitis and epididymitis in men ( CDC, 2020 ; Control ECfDPa, 2022 ). However, Ct urogenital infections are notorious for being asymptomatic or sub-clinically symptomatic in as many as 70% of women and 50% of men ( CDC, 2020 ; Gupta et al., 2021 ; Control ECfDPa, 2022 ). These “silent” Ct infections are often left untreated, which can last for months to years ( McCormack et al., 1979 ; Morré et al., 2002 ; Molano et al., 2005 ; Geisler, 2010 ). Approximately 17% of Ct -infected women go on to develop pelvic inflammatory disease (PID) and serious complications such as tubal factor infertility (TFI), ectopic pregnancy and chronic pelvic pain ( Cherpes et al., 2011 ; Brunham et al., 2015 ; Price et al., 2016 ). Repeated Ct infections and recurrent PID episodes are associated with a greater risk for adverse reproductive outcomes ( Hillis et al., 1997 ; Hosenfeld et al., 2009 ; Batteiger et al., 2010 ; Trent et al., 2011 ; Bautista et al., 2018 ). Epidemiological evidence indicate that Ct infections significantly increase the risk of cervical ( Zhu et al., 2016 ) and ovarian cancer ( Hosseininasab-Nodoushan et al., 2021 ), presumably due to unresolved chronic inflammation ( Clendenen et al., 2011 ; Peres et al., 2021 ). While considerable efforts have been made in attempts to elucidate Ct pathogenesis, there are still gaps regarding the definitive molecular and cellular mechanisms that cause Ct -associated tissue damage. Chlamydia species have a unique biphasic lifecycle consisting of an extracellular form of non-replicative but infectious elementary body (EB), and an intracellular form of replicative reticulate body (RB) ( Moulder, 1991 ; Hackstadt et al., 1997 ). Infection is normally initiated at the single layer of mucosal epithelium in the lower genital tract, followed by bacterial ascension to the upper genital tract. Once inside the host cells, the non-replicative EB resides within a cytoplasmic inclusion body, where it differentiates into a replicative RB which then multiplies via binary fission. As an inclusion fills with progeny, RBs transform back into infectious EBs that are released from host cells to infect other neighboring cells ( Moulder, 1991 ; Hackstadt et al., 1997 ). Immediately after intracellular infection, cell-autonomous immunity and innate defence mechanisms are triggered to fight against intracellular Ct infections ( Mostowy and Shenoy, 2015 ; Elwell et al., 2016 ). These initial molecular and cellular events are followed by induction of Ct -specific adaptive humoral and cellular immune responses that are required for the ultimate elimination of intracellular Ct infection ( Kelly, 2003 ; Batteiger et al., 2010 ; Helble and Starnbach, 2021 ). Notably, the inflammatory responses associated with innate and adaptive immunity are also involved in the tissue repair and regeneration processes ( Eming et al., 2017 ), which are critical for removing the cellular debris from the infected tissue site, maintaining the tissue homeostasis, and restoring the normal structure and physiological integrity of the organ after infection ( Gurtner et al., 2008 ). Of note, Chlamydia species are known for their strong ability to exploit various strategies to enhance intracellular survival ( Fischer and Rudel, 2018 ; Chen et al., 2019 ). Some RBs ultimately differentiate into a morphologically distinct persistence form called an aberrant body (AB) in response to host defense molecules and certain antibiotic treatments ( Phillips et al., 1984 ; Mazzoli et al., 2000 ; Hogan et al., 2004 ). While ABs are nonreplicative, they remain dormant inside host cells for a long period of time and are capable of reverting back to replicative RBs when more favorable conditions are present, such as the use of an immune suppressant ( Yang et al., 1983 ; Beatty et al., 1994 ; Laitinen et al., 1996 ). As such, the host responses to Ct infection are complex and often modified by persistent infection, reactivation, and/or repeated exposures ( Schuchardt and Rupp, 2018 ). The most widely accepted theory of Ct pathogenesis is that unresolved chronic inflammation is the primary culprit of Ct -associated tissue damage. This involves aberrant production of cytokines, chemokines, and growth factors, as well as the influx of innate and adaptive immune cells that collectively trigger aggravated wound healing and tissue repair processes ( Karin and Clevers, 2016 ). While numerous cytokines have been implicated as part of the host response to Ct infection ( Yang, 2003 ; Xiang et al., 2021 ), leukemia inhibitory factor (LIF), a member of the interleukin 6 (IL-6) cytokine family, has been largely ignored. Recent studies indicate that LIF is readily induced by Ct infection ( Hess et al., 2001 ; Kessler et al., 2019 ), and that increased LIF expression is often linked to Ct -associated ectopic pregnancies in humans ( Refaat et al., 2016 ) as well as severe oviductal pathology in mice ( Hou et al., 2018 ). Therefore, LIF likely holds new perspectives of Ct pathogenesis. In this article, we review the biology of LIF and LIF receptor (LIFR)-mediated signaling pathways, summarize the physiological role of LIF in the reproductive system, and discuss the impact of LIF in chronic inflammatory conditions and its implication in Ct pathogenesis.

In conclusion, available evidence supports a novel aspect of Ct pathogenesis controlled by the pleiotropic cytokine LIF ( Figure 4 ). Despite the intended purpose of LIF production as a part of the host defense against intracellular Ct infection and host protective tissue healing, LIF-mediated signaling, particularly prolonged strong signaling, gradually transforms the microenvironment of the fallopian tube by diminishing the density of ciliated epithelial cells and increasing the population of less differentiated secretory epithelial cells. This harmful transformation of epithelium might be a key process leading to an enhanced risk of infertility, ectopic pregnancy and cancer. A potential role of LIF in Chlamydia pathogenesis. LIF is produced by epithelial cells upon Ct infection, which is required for maintaining epithelium homeostasis and normal tissue repair via autocrine LIF/LIFR signaling pathways. This would occur in many Ct- infected women without leaving serious complications. However, aberrant tissue repair is triggered by repeated or persistent infections, which results in marked reductions of ciliated epithelial cells and increases in secretory cells over time. The alteration in the cellular composition creates a microenvironment in fallopian tubes that may promote infertility, ectopic pregnancy and cancer.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The study is supported by operating grant of the Canadian Institute of Health Research (CIHR) (Grant number: 201803PJT-159700) to JW. KW is supported by a Dalhousie Medical Research Foundation (DMRF) – Infection, Immunity, Inflammation & Vaccinology (I3V) Graduate Studentship.

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.