Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric myelodysplastic syndrome or myeloproliferative disorder for which hematopoietic stem cell transplantation remains the only curative option; however, outcomes are particularly poor in patients harboring PTPN11 (encodes SHP2 phosphatase) mutations. Using a Shp2E76K/+ JMML mouse model, we identify a pathogenic IL-17A/PTGS2/NLRP3 signaling axis that drives bone marrow inflammation, suppresses antitumor immunity, and promotes leukemic progression. Shp2E76K/+ mice exhibited profound immune dysregulation, characterized by expansion of regulatory T cells (Tregs), increased T-cell exhaustion, and impaired cytotoxic function with reduced CD4⁺ and CD8⁺ T-cell frequencies. Mechanistically, mutant macrophages upregulated IL-17A, triggering NLRP3 inflammasome activation, PTGS2 induction, caspase-1 cleavage, and IL-1β maturation, thereby amplifying inflammatory signaling within the marrow niche. Therapeutically, IL-17A neutralization suppressed inflammasome activity, while combined inhibition of NLRP3 and PTGS2 restored cytotoxic T-cell function, reduced systemic and marrow inflammation, reversed myeloproliferation, and significantly prolonged survival in Shp2E76K/+ mice. Importantly, ex vivo treatment of primary JMML patient samples with dual NLRP3/PTGS2 inhibition combined with MEK blockade significantly reduced leukemic progenitor colony formation, supporting translational relevance. In patient-derived xenograft models of PTPN11-mutant JMML, dual NLRP3/PTGS2 inhibition combined with MEK blockade most effectively reduced leukemic burden, decreased human CD45⁺ engraftment, and depleted leukemic CD34⁺CD38⁺ progenitors and GMPs while restoring MEP populations, resulting in significantly improved overall survival. Together, these findings establish IL-17A/PTGS2/NLRP3 signaling as a central driver of immune suppression and myeloid expansion in PTPN11-mutant JMML and highlight combinatorial anti-inflammatory targeting as a promising therapeutic strategy for this high-risk disease.

Obesity is increasingly implicated in hematopoietic malignancies, yet its role in mutation-driven myeloid leukemias remains unclear. Using UK Biobank data from over 440,000 individuals, we found obesity traits including elevated BMI and waist-to-hip ratio were associated with type 2 diabetes, increased plasma IL-17A levels, reduced glucagon-like peptide 1 receptor (GLP-1R) expression, and heightened risk of myeloid malignancies. Transplantation of protein tyrosine phosphatase nonreceptor type 11 (PTPN11) (Shp2E76K/+) mutant hematopoietic stem/progenitor cells into obese mice demonstrated that metabolic inflammation accelerated leukemogenesis via myeloid cell expansion, lipid metabolic rewiring, IL-17A activation, and accumulation of M2-like tumor-associated macrophages (TAMs), accompanied by T cell exhaustion and impaired antigen presentation. Notably, dual therapy with an anti-IL-17A antibody and a GLP-1R agonist reversed these effects by reducing M2-like TAMs, restoring Ciita-dependent antigen presentation and Tyk2-mediated IFN-γ signaling, reactivating T cell responses, and reducing leukemic burden. These findings establish IL-17A-driven, metabolism-coupled immunosuppression as a mechanistic link between obesity and protein tyrosine phosphatase 2-mutant (SHP2-mutant) myeloid leukemias, highlighting a tractable therapeutic strategy for patients with obesity at high risk for other diseases and their complications.

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by age-related somatic mutations in hematopoietic stem and progenitor cells (HSC/Ps) and is correlated with an increased risk of myeloid malignancies, elevated inflammatory pathways in circulating myeloid cells, higher all-cause mortality, chronic kidney disease, and cardiovascular disease. The pathophysiology of inflammatory bowel disease (IBD) is intrinsically linked to heightened inflammation. Nevertheless, the presence of CHIP in IBD and its role in the pathophysiology of IBD remains poorly elucidated. In the UK Biobank, CHIP was associated with an increased incidence of IBD. Females with CHIP had a 1.33-fold higher risk, which was further validated in All of Us data base (ßOR = 1.29). For Crohn’s disease, DNMT3A mutations conferred a 1.81-fold increased incidence in females compared to non-DNMT3A-carriers, which rose to 2.09 for large clones (variant allele fraction ≥10%). In contrast, for ulcerative colitis, TET2 large clones were significantly associated, and only among individuals under 45. These associations were further identified using two-sample Mendelian randomization. In a mouse model of CHIP-IBD, HSC/Ps with Dnmt3a mutation demonstrated significantly worse pathophysiology compared to controls, due in part to heightened expression of Apurinic/apyrimidinic endonuclease 1 (APE1) in the bone marrow and colon. Treatment with the APE1/Ref-1 inhibitor APX3330 ameliorated CHIP-IBD driven by the Dnmt3a mutation.

Abstract Ulcerative colitis (UC) is a debilitating, immune-mediated inflammatory disorder of the gastrointestinal (GI) tract with far-reaching consequences on distal organs, including the bone marrow. Here, we describe the molecular mechanisms that contribute to UC-induced abnormal hematopoiesis. We show that chronic UC drives HSPC differentiation toward myelopoiesis in an APE1/Ref-1/HIF-1α/IL-1r1-dependent manner. Blockade of the redox-activity of APE1/Ref-1 with APX3330 inhibits the elevated expression of HIF-1α in HSPCs and reverses the aberrant HSPC dynamics under the inflammatory milieu of UC, including suppression of pro-inflammatory Ly6Chi monocytes. Using echinomycin, we pharmacologically blocked HIF-1α activity and found that HIF-1α mediates inflammatory responses via downstream IL-1r1 signaling. Blockade of the redox activity of ref-1 rescues the abnormal HSPC function. Our data highlight the significance of the APE1/Ref-1/HIF-1α/IL-1r1 signaling cascade in aberrant hematopoiesis that contributes to the pathophysiology of chronic UC through a feed-forward loop.

Chlamydia trachomatis infection is the most common bacterial sexually transmitted infection worldwide and a leading cause of inflammatory reproductive tract disease and infertility in women. Much of the tissue damage associated with genital chlamydial infection arises from host inflammatory responses rather than direct bacterial cytotoxicity. Epithelial cells lining the female reproductive tract represent the primary host cells infected during chlamydial infection and play key roles in initiating innate immune responses. Among the cytokines produced by infected epithelial cells, type-I interferons have emerged as important regulators of host defense and inflammatory signaling; however, the specific contribution of interferon-β (IFN-β) to epithelial transcriptional responses during chlamydial infection remains incompletely defined. In the present study, we investigated the role of IFN-β in coordinating epithelial immune signaling networks during infection with Chlamydia muridarum. Using wild-type murine oviduct epithelial cells (OE-WT) and IFN-β-deficient epithelial cells (OE-IFNβ-KO), we performed pathway-focused RT2 Profiler PCR array analyses examining transcriptional responses across four biological pathways: (1) innate and adaptive immune responses, (2) type-I interferon signaling, (3) inflammatory and autoimmune responses, and (4) fibrosis-associated pathways. Infection of OE-WT cells resulted in coordinated induction of cytokines, chemokines, and interferon-stimulated genes associated with antimicrobial defense and immune cell recruitment. In contrast, IFN-β deficiency resulted in widespread dysregulation of these transcriptional programs, including reduced induction of interferon-responsive chemokines such as CCL5 and CXCL10, altered inflammatory cytokine expression, and transcriptional signatures consistent with enhanced tissue remodeling responses. Notably, IFN-β deficiency resulted in increased TNF expression accompanied by reduced IL-6 induction, suggesting disruption of balanced inflammatory signaling networks. Pathway analyses further revealed dysregulated expression of fibrosis-associated genes including Serpine1, Ctgf, and Eng in IFN-β-deficient epithelial cells, indicating potential mechanisms linking interferon signaling to tissue remodeling during infection. Collectively, these findings identify IFN-β as a central regulator of epithelial immune networks during chlamydial infection and suggest that disruption of IFN-β signaling may promote inflammatory and fibrotic pathology within the female reproductive tract.

Chlamydia trachomatis infection of the female genital tract can result in severe reproductive sequelae, including pelvic inflammatory disease, tubal scarring, and infertility. Type I interferons have been implicated in both host defense and immunopathogenesis during chlamydial infection, with conflicting conclusions across experimental systems. However, the specific contributions of individual interferon subtypes remain poorly defined. Here, we examined the role of interferon beta (IFN-β) in regulating epithelial immune responses and intracellular bacterial development during Chlamydia muridarum infection. Using murine oviduct epithelial (OE) cell lines derived from wild-type, IFNβ-deficient, and Toll-like receptor 3 (TLR3)-deficient mice, we demonstrate that IFN-β is a critical epithelial-intrinsic mediator of host defense. Loss of IFN-β led to dysregulation of genes associated with inflammation, immune regulation, and fibrosis, altered chlamydial inclusion morphology, enhanced expression of bacterial genes throughout the chlamydial developmental cycle, and increased chlamydial replication. Importantly, exogenous IFN-β restored both immune mediator production and bacterial control during IFNβ-deficiency. Parallel analyses revealed that TLR3 deficiency phenocopied IFN-β loss, supporting a TLR3-IFN-β signaling axis that restricts chlamydial growth. Consistent with these in vitro findings, IFNβ-deficient mice exhibited enhanced bacterial burden during genital tract infection. Together, these data establish IFN-β as a protective epithelial mediator during chlamydial infection and demonstrate that type I interferon responses are not functionally uniform. Our findings provide a mechanistic framework to reconcile the protective role of IFN-β with reports of reduced pathology in interferon-α/β receptor-deficient models and highlight the importance of dissecting individual interferon pathways in chlamydial immunopathogenesis. Importance:Genital tract infection with Chlamydia trachomatis remains a leading cause of preventable infertility worldwide. Although type I interferons are widely viewed as contributors to chlamydial pathology, most studies have examined global interferon signaling rather than the roles of individual interferon subtypes. In this study, we demonstrate that interferon beta (IFN-β) plays a protective, epithelial-intrinsic role during chlamydial infection by restricting bacterial development and shaping local immune responses. These findings challenge the prevailing view that type I interferons are uniformly detrimental in this setting and reveal that distinct interferon subtypes can exert opposing effects on host defense and disease outcome. By defining a TLR3-IFN-β signaling axis that limits chlamydial replication, this work advances our understanding of epithelial immunity in the female genital tract and has important implications for the design of targeted immunomodulatory strategies to prevent chlamydia-induced reproductive pathology.

Bone marrow (BM) is a tissue that is of great importance to several areas of basic and translational research, including hematology, oncology, bone biology, and immunology. It is unique in that it is gelatinous in nature but housed in a hard casing of bone. Traditionally, flow cytometry and immunofluorescence (IF) techniques have been employed to study the composition of cellular interactions and elements of the BM. However, it has been challenging to study the BM in an unperturbed state using multiple fluorescent probes at a time to fully appreciate the diverse cell populations and their interactions and relative positioning with each other. This protocol addresses how Phenocycler 2.0TM, which uses co-detection by indexing (CODEX) in conjunction with HALO 4.0TM image analysis software, can overcome the obstacles faced by traditional techniques used to study the BM in an unperturbed state.

Ex vivo expansion of hematopoietic stem cells (HSCs) is limited by mitochondrial stress-induced loss of stemness. To identify protective mechanisms resembling the hypoxic bone marrow niche, we performed single-cell transcriptomics on hypoxia-collected HSCs, revealing significant downregulation of Rho-associated kinase 1 (ROCK1). This observation suggested ROCK1 as a potential regulator of HSC homeostasis. We tested this by genetically or pharmacologically inhibiting ROCK1 with shRNA or Y27632, which reduced mitochondrial reactive oxygen species, mitochondrial mass, and membrane potential while promoting expansion of phenotypic HSCs (Lin⁻CD34⁺CD38⁻CD45RA⁻CD49f⁺CD90⁺). Mechanistically, ROCK1 inhibition attenuated DRP1-mediated mitochondrial fission by decreasing p-DRP1(Ser616) and increasing p-DRP1(Ser637), thereby reducing mitochondrial fragmentation. Additionally, ROCK1 inhibition elevated BCL2 expression and reduced active Caspase-3 levels, indicating suppressed apoptosis. Limiting dilution transplants demonstrated a fourfold increase in functional HSC frequency following ROCK1 inhibition, with enhanced long-term engraftment in secondary recipients. Our findings identify ROCK1 as a critical regulator of mitochondrial dynamics in HSCs and provide a mechanistic basis for targeting ROCK1 to enhance functional HSC expansion, offering a promising strategy to improve outcomes in HSC transplantation by mimicking hypoxic niche signals ex vivo.

Acute myeloid leukemia (AML) patients present with CD8 exhaustion signatures, and pharmacologic inhibition of checkpoints can have therapeutic benefit. The alarmin IL-33 and its receptor STimulation-2 (ST2) promote activation of tissue-regulatory T cells (Treg cells) and accelerate malignant progression in solid tumors, but their role in leukemia remains unclear. Here, we show that ST2+ Treg cells are enriched in bone marrow (BM) of humans and mice with AML and promote CD8+ T cells depletion and exhaustion. ST2 deficiency in Treg cells restores CD8+ T cell function, decreasing AML growth via retention of ST2+ Treg cells precursors in lymph nodes. AML-activated ST2+ Treg cells lack T-bet, IFN-γ and Bcl-6, and kill intratumoral CD8+ T cells by amplified granzyme B-mediated cytotoxicity compared to non-AML primed Treg cells. Engineered anti-ST2 antibodies induce ST2+ Treg cells apoptosis to extend survival in AML models. Together, our findings suggest that ST2 is a potential checkpoint target for AML immunotherapy.

Characterized by the accumulation of somatic mutations in blood cell lineages, clonal hematopoiesis of indeterminate potential (CHIP) is frequent in aging and involves the expansion of mutated hematopoietic stem and progenitor cells (HSC/Ps) that leads to an increased risk of hematologic malignancy. However, the risk factors that contribute to CHIP-associated clonal hematopoiesis (CH) are poorly understood. Obesity induces a proinflammatory state and fatty bone marrow (FBM), which may influence CHIP-associated pathologies. We analyzed exome sequencing and clinical data for 47,466 individuals with validated CHIP in the UK Biobank. CHIP was present in 5.8% of the study population and was associated with a significant increase in the waist-to-hip ratio (WHR). Mouse models of obesity and CHIP driven by heterozygosity of Tet2, Dnmt3a, Asxl1, and Jak2 resulted in exacerbated expansion of mutant HSC/Ps due in part to excessive inflammation. Our results show that obesity is highly associated with CHIP and that a proinflammatory state could potentiate the progression of CHIP to more significant hematologic neoplasia. The calcium channel blockers nifedipine and SKF-96365, either alone or in combination with metformin, MCC950, or anakinra (IL-1 receptor antagonist), suppressed the growth of mutant CHIP cells and partially restored normal hematopoiesis. Targeting CHIP-mutant cells with these drugs could be a potential therapeutic approach to treat CH and its associated abnormalities in individuals with obesity.

Summary Background Type I IFN (IFN-I) is a family of cytokines involved in the pathogenesis of autoimmune and autoinflammatory diseases such as psoriasis. SIDT1 is an ER-resident protein expressed in the lymphoid lineage, and involved in anti-viral IFN-I responses in vivo, through an unclear mechanism. Herein we have dissected the role of SIDT1 in the natural IFN-producing cells, the plasmacytoid dendritic cells (pDC). Methods The function of SIDT1 in pDC was determined by silencing its expression in human primary pDC and GEN2.2 cell line. SIDT1 role in vivo was assessed using the imiquimod-induced psoriasis model in the SIDT1-deficient mice (sidt1-/-). Findings Silencing of SIDT1 in GEN2.2 led to a blockade of the IFN-I response after stimulation of TLR7 and TLR9, without affecting the pro-inflammatory responses or upregulation of maturation markers. We found that SIDT1 migrates from the ER to the endosomal and lysosomal compartments together with TLR9 after CpG stimulation, participating in the access of the TLR9-CpG complex to lysosome-related vesicles, and therefore mediating the activation of TBK1 and the nuclear migration of IRF7, but not of NF -KB. sidt1-/- mice showed a significant decrease in severity parameters of the imiquimod-induced acute psoriasis-like model, associated with a decrease in the production of IFN-I and IFN-dependent chemokines. Interpretation Our findings indicate that SIDT1 is at the cross-road between the IFN-I and the proinflammatory pathways and constitutes a promising drug target for psoriasis and other diseases mediated by IFN-I responses. Funding This work was supported by the Consejer?a de Salud y Familias de la Junta de Andaluc?a (PIER_S1149 and C2_S0050) and Instituto de Salud Carlos III (PI18/00082 and PI21/01151), partly supported by European FEDER funds, and prior funding to MEAR from the Alliance for Lupus Research and the Swedish Research Council. Copyright (c) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) eBioMedicine 103808 Published uary https://doi.org/10.1016/j. ebiom.2021.103808 Superscript/Subscript Available

Abstract Obesity is an increasing epidemic world-wide responsible for enhancing the risk for developing Type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD) and cancer. However, it is unclear if and how obesity contributes to the transformation of pre-leukemic stem and progenitors (pre-LHSC/Ps) into full-blown leukemia such as acute myeloid leukemia (AML) or severe form of myeloproliferative neoplasm (MPN) or CVD. We hypothesized that obesity induced chronic inflammation might be responsible for clonal selection of pre-LHSC/Ps bearing pre-leukemic clonal hematopoiesis of indeterminate potential (CHIP) mutations such as DNMT3A, TET2, ASXL1, and JAK2 and for promoting the progression of early-onset MPN, AML/leukemia and CVD. To study the linkage between obesity and CHIP in humans, we first examined the UK biobank. After exclusions, the final study cohort included 47,466 unrelated participants free of T2DM at baseline and having valid CHIP measurements. The mean (SD) age at enrollment was 56.5 (8.0), 45.0% were male, 43.9% never smoked, and 82.6% self-reported as European decedents. At baseline, the mean (SD) body mass index (BMI) was 27.3 (4.7) kg/m 2, with 43.0% overweight and 23.6% obese, and the overall mean (SD) waist-to-hip ratio (WHR) was 0.87 (0.09). CHIP was present among 5.8% of the study population the most common mutations on the DNMT3A (3.7%) and TET2 (1.0%) genes; large CHIP clone defined as CHIP mutation with variant allele fraction >10% was present among 2.4% of the study population. Individuals with CHIP mutations on average had higher WHR. The presence of CHIP mutation was associated with a 0.0028 increase of WHR (p=0.03). Furthermore, CHIP prevalence increased with higher WHR: the percentage of participants with CHIP was 4.93%, 5.75%, 6.56% in the lowest, middle, and highest WHR quintiles respectively, signifying that dysfunctional metabolism may accelerate expansion of clonal hematopoiesis (CH). To better define the mechanism of obesity driven CH, we utilized several novel mouse models bearing Tet2 -/-, Dnmt3a +/-, Asxl1 +/- and Jak2 +/- mutations to mimic the human pre-LHSC/Ps condition and obesity, in the form of leptin deficient Lep Ob/Ob (Ob/Ob) mutation, which induces obesity and T2DM. We show that both the compound mutant (Tet2 -/-;Ob/Ob, Dnmt3a +/-;Ob/Ob, Asxl1 +/-;Ob/Ob and Jak2 +/-;Ob/Ob) and CHIP mutant bone marrow (BM; Tet2 -/-, Dnmt3a +/-, Asxl1 +/- and Jak2 +/-) transplanted into Ob/Ob mice develop rapid growth of mature myeloid cells and HSC/Ps leading to severe form of MPN/AML as well as CVD. This was associated with upregulation of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. Flow cytometry analysis of LSK and progenitor cells isolated from Tet2 -/-;Ob/Ob mice revealed an up-regulation of intracellular Ca2+ levels. We hypothesized that up-regulated Ca2+ signaling in Tet2 -/-; Ob/Ob HSC/Ps promotes aberrant signaling leading to an early-onset of severe MPN/AML. We performed a competitive transplantation experiment using, Tet2 -/-: Boy/J BM cells (1:1 ratio) into Ob/Ob and WT recipients. After 8 weeks post transplantation, we investigated the role of Ca2+ blockers in driving CH in Ob/Ob recipients using pharmacological inhibitors, either individually, or in combination, of metformin (100 mg/kg, orally), nifedipine (100 mg/kg, orally), MCC950 (30 mg/kg, orally) and anakinra (10 mg/kg, i.p). The combination treatment markedly reduced monocytes, neutrophils, WBC counts, and improved RBCs, hematocrits and platelets. The spleen and liver, heart, body weights and blood glucose levels were significantly reduced, along with a greater re-emergence of normal CD45.1 wild-type cells in the PB, BM, and spleen and a significant reduction in Tet2 mutant CD45.2 pre-LHSC/Ps and myeloid cells in the PB, BM, and spleen. Importantly, the frequency of leukemic blasts, LSK cells, ST-HSCs, LT-HSCs and granulocyte macrophage progenitors (GMP) were significantly reduced. Furthermore, the combination of drug treatment showed greater heart protective activity by reducing the atherosclerotic lesions in Ob/Ob recipients bearing CHIP by suppressing Ca2+ signaling. Taken together, these data suggest that obesity is highly associated with the presence of CHIP in humans and that targeting CHIP mutant cells with a combination of metformin/nifedipine/MCC950/anakinra is a safe and inexpensive way to rescue CH and its associated leukemic and cardiovascular defects. Disclosures Natarajan: Amgen: Research Funding; Apple: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Boston Scientific: Research Funding; Blackstone Life Sciences: Consultancy; Genentech: Consultancy; Foresite Labs: Consultancy.

Reproductive tract pathology caused by Chlamydia trachomatis infection is an important global cause of human infertility. To better understand the mechanisms associated with Chlamydia-induced genital tract pathogenesis in humans, we used CRISPR genome editing to disrupt Toll-like receptor 3 (TLR3) function in the human oviduct epithelial (hOE) cell line OE-E6/E7 in order to investigate the possible role(s) of TLR3 signaling in the immune response to Chlamydia Disruption of TLR3 function in these cells significantly diminished the Chlamydia-induced synthesis of several inflammation biomarkers, including interferon beta (IFN-β), interleukin-6 (IL-6), interleukin-6 receptor alpha (IL-6Rα), soluble interleukin-6 receptor beta (sIL-6Rβ, or gp130), IL-8, IL-20, IL-26, IL-34, soluble tumor necrosis factor receptor 1 (sTNF-R1), tumor necrosis factor ligand superfamily member 13B (TNFSF13B), matrix metalloproteinase 1 (MMP-1), MMP-2, and MMP-3. In contrast, the Chlamydia-induced synthesis of CCL5, IL-29 (IFN-λ1), and IL-28A (IFN-λ2) was significantly increased in TLR3-deficient hOE cells compared to their wild-type counterparts. Our results indicate a role for TLR3 signaling in limiting the genital tract fibrosis, scarring, and chronic inflammation often associated with human chlamydial disease. Interestingly, we saw that Chlamydia infection induced the production of biomarkers associated with persistence, tumor metastasis, and autoimmunity, such as soluble CD163 (sCD163), chitinase-3-like protein 1, osteopontin, and pentraxin-3, in hOE cells; however, their expression levels were significantly dysregulated in TLR3-deficient hOE cells. Finally, we demonstrate using hOE cells that TLR3 deficiency resulted in an increased amount of chlamydial lipopolysaccharide (LPS) within Chlamydia inclusions, which is suggestive that TLR3 deficiency leads to enhanced chlamydial replication and possibly increased genital tract pathogenesis during human infection.

PROBLEM:Chlamydia trachomatis infections are often associated with acute syndromes including cervicitis, urethritis, and endometritis, which can lead to chronic sequelae such as pelvic inflammatory disease (PID), chronic pelvic pain, ectopic pregnancy, and tubal infertility. As epithelial cells are the primary cell type productively infected during genital tract Chlamydia infections, we investigated whether Chlamydia has any impact on the integrity of the host epithelial barrier as a possible mechanism to facilitate the dissemination of infection, and examined whether TLR3 function modulates its impact.METHOD OF STUDY:We used wild-type and TLR3-deficient murine oviduct epithelial (OE) cells to ascertain whether C. muridarum infection had any effect on the epithelial barrier integrity of these cells as measured by transepithelial resistance (TER) and cell permeability assays. We next assessed whether infection impacted the transcription and protein function of the cellular tight-junction (TJ) genes for claudins1-4, ZO-1, JAM1 and occludin via quantitative real-time PCR (qPCR) and western blot.RESULTS:qPCR, immunoblotting, transwell permeability assays, and TER studies show that Chlamydia compromises cellular TJ function throughout infection in murine OE cells and that TLR3 deficiency significantly exacerbates this effect.CONCLUSION:Our data show that TLR3 plays a role in modulating epithelial barrier function during Chlamydia infection of epithelial cells lining the genital tract. These findings propose a role for TLR3 signaling in maintaining the integrity of epithelial barrier function during genital tract Chlamydia infection, a function that we hypothesize is important in helping limit the chlamydial spread and subsequent genital tract pathology.

ABSTRACTReproductive tract pathology caused byChlamydia trachomatisinfection is an important global cause of human infertility. To better understand the mechanisms associated withChlamydia-induced genital tract pathogenesis in humans, we used CRISPR genome editing to disrupt TLR3 function in the human oviduct epithelial (hOE) cell-line OE-E6/E7, in order to investigate the possible role(s) of TLR3 signaling in the immune response toChlamydia. Disruption of TLR3 function in these cells significantly diminished theChlamydia-induced synthesis of several inflammation biomarkers including IFN-β, IL-6, IL-6Ra, sIL-6Rβ (gp130), IL-8, IL-20, IL-26, IL-34, sTNF-R1, TNFSF13B, MMP-1, MMP-2, and MMP-3. In contrast, theChlamydia-induced synthesis of CCL-5, IL-29 (IFNλ1) and IL-28A (IFNλ2) were significantlyincreasedin the TLR3-deficient hOE cells when compared to their wild-type counterparts. Our results propose a role for TLR3 signaling in limiting the genital tract fibrosis, scarring, and chronic inflammation often associated with human chlamydial disease. Interestingly, we saw thatChlamydiainfection induced the production of biomarkers associated with persistence, tumor metastasis, and autoimmunity such as soluble CD163 (sCD163), chitinase-3-like protein 1, osteopontin, and pentraxin-3 in the hOE cells; however, their expression levels were significantly dysregulated in the TLR3-deficient hOE cells. Finally, we demonstrate using the hOE cells that TLR3 deficiency resulted in an increased amount of chlamydial LPS within theChlamydiainclusion, which is suggestive that TLR3 deficiency leads to enhanced chlamydial replication and possibly increased genital tract pathogenesis during human infection.AbbreviationshOE, human OE-E6/E7 cells; TLR3 KO, TLR3 knockout cell line; poly (I:C), Polyinosinic–polycytidylic acid sodium salt.

Chlamydia trachomatis replicates primarily in the epithelial cells lining the genital tract and induces the innate immune response by triggering cellular pathogen recognition receptors (PRRs).Our previous studies showed that Toll-like receptor 3 (TLR3) is expressed in murine oviduct epithelial (OE) cells, is the primary PRR triggered by C. muridarum (Cm) early during infection to induce IFN-β synthesis, and that TLR3 signaling regulates the chlamydial induced synthesis of a plethora of other innate inflammatory modulators including IL-6, CXCL10, CXCL16 and CCL5.We also showed that the expression of these cytokines induced by Chlamydia was severely diminished during TLR3 deficiency; however, the replication of Chlamydiain TLR3 deficient OE cells was more robust than in WT cells.These data suggested that TLR3 had a biological impact on the inflammatory response to Chlamydia infection; however, the global effects of TLR3 signaling in the cellular response to Chlamydia infection in murine OE cells has not yet been investigated.To determine the impact of TLR3 signaling on Chlamydia infection in OE cell at the transcriptome level, we infected wild-type (OE-WT) and TLR3-deficient (OE-TLR3KO) cells with Cm, and performed transcriptome analyses using microarray.Genome-wide expression and ingenuity pathway analysis (IPA) identified enhanced expression of host genes encoding for components found in multiple cellular processes encompassing: (1) pro-inflammatory, (2) cell adhesion, (3) chemoattraction, (4) cellular matrix and small molecule transport, (5) apoptosis, and (6) antigen-processing and presentation.These results support a role for TLR3 in modulating the host cellular responses to Cm infection that extend beyond inflammation and fibrosis, and shows that TLR3 could serve a potential therapeutic target for drug and/or vaccine development.

Chlamydia trachomatis urogenital serovars primarily replicate in epithelial cells lining the reproductive tract. Epithelial cells recognize Chlamydia through cell surface and cytosolic receptors, and/or endosomal innate receptors such as Toll-like receptors (TLRs). Activation of these receptors triggers both innate and adaptive immune mechanisms that are required for chlamydial clearance, but are also responsible for the immunopathology in the reproductive tract. We previously demonstrated that Chlamydia muridarum (Cm) induces IFN-β in oviduct epithelial cells (OE) in a TLR3-dependent manner, and that the synthesis of several cytokines and chemokines are diminished in Cm-challenged OE derived from TLR3-/- 129S1 mice. Furthermore, our in vitro studies showed that Cm replication in TLR3-/- OE is more efficient than in wild-type OE. Because TLR3 modulates the release inflammatory mediators involved in host defense during Cm infection, we hypothesized that TLR3 plays a protective role against Cm-induced genital tract pathology in congenic C57BL/6N mice. Using the Cm mouse model for human Chlamydia genital tract infections, we demonstrated that TLR3-/- mice had increased Cm shedding during early and mid-stage genital infection. In early stage infection, TLR3-/- mice showed a diminished synthesis of IFN-β, IL-1β, and IL-6, but enhanced production of IL-10, TNF-α, and IFN-γ. In mid-stage infection, TLR3-/- mice exhibited significantly enhanced lymphocytic endometritis and salpingitis than wild-type mice. These lymphocytes were predominantly scattered along the endometrial stroma and the associated smooth muscle, and the lamina propria supporting the oviducts. Surprisingly, our data show that CD4+ T-cells are significantly enhanced in the genital tract TLR3-/- mice during mid-stage Chlamydial infection. In late-stage infections, both mouse strains developed hydrosalpinx; however, the extent of hydrosalpinx was more severe in TLR3-/- mice. Together, these data suggest that TLR3 promotes the clearance of Cm during early and mid-stages of genital tract infection, and that loss of TLR3 is detrimental in the development hydrosalpinx.

The electrophoresis on cellulose acetate membrane is most widely used because of its simplicity, and is without the use of any sophisticated instrument other than electrophoresis apparatus and the cellulose acetate strip. Here we describe a modified version of cellulose acetate membrane electrophoresis for hemoglobin separation from blood sample. Sharp, clear bands without tailing effects can be obtained with this method. The method and apparatus described here would be appropriate to separate protein fractions under 1 h at voltages up to 60 V/cm measured between the electrodes.

The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1-/- mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. Bank1 deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from Bank1 deficient mice had strongly reduced Ifnb, Ifna4, Irf7, Aicda and Stat1 gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1-/- mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease.