Transcriptome and Functions of Granulocytic Myeloid-Derived Suppressor Cells Determine their Association with Disease Severity of COVID-19.

COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19, that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of Granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1 + G-MDSC (Arg + G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg + G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.

Presence of antibody-dependent cellular cytotoxicity (ADCC) against SARS-CoV-2 in COVID-19 plasma.

BACKGROUND: Neutralizing-antibody (nAb) is the major focus of most ongoing COVID-19 vaccine trials. However, nAb response against SARS-CoV-2, when present, decays rapidly. Given the myriad roles of antibodies in immune responses, it is possible that antibodies could also mediate protection against SARS-CoV-2 via effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC), which we sought to explore here. METHODS: Plasma of 3 uninfected controls and 20 subjects exposed to, or recovering from, SARS-CoV-2 infection were collected from U.S. and sub-Saharan Africa. Immunofluorescence assay was used to detect the presence of SARS-CoV-2 specific IgG antibodies in the plasma samples. SARS-CoV-2 specific neutralizing capability of these plasmas was assessed with SARS-CoV-2 spike pseudotyped virus. ADCC activity was assessed with a calcein release assay. RESULTS: SARS-CoV-2 specific IgG antibodies were detected in all COVID-19 subjects studied. All but three COVID-19 subjects contained nAb at high potency (>80% neutralization). Plasma from 19/20 of COVID-19 subjects also demonstrated strong ADCC activity against SARS-CoV-2 spike glycoprotein, including two individuals without nAb against SARS-CoV-2. CONCLUSION: Both neutralizing and non-neutralizing COVID-19 plasmas can mediate ADCC. Our findings argue that evaluation of potential vaccines against SARS-CoV-2 should include investigation of the magnitude and durability of ADCC, in addition to nAb.

APOL1 Risk Variants and Acute Kidney Injury in Black Americans with COVID-19.

BACKGROUND AND OBJECTIVES: Black Americans have a higher incidence of kidney disease compared with populations that do not have recent African ancestry. Two risk variants in the APOL1 are responsible for a portion of this higher risk. We sought to assess the odds of AKI conferred by APOL1 risk alleles in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Black Americans who tested positive for coronavirus disease 2019 (COVID-19) were genotyped to determine APOL1 risk allele status. We assessed the incidence of AKI, persistent AKI, and AKI requiring KRT within 21 days of the PCR-based diagnosis. Outcomes were adjusted for age, sex, body mass index, hypertension, eGFR, and use of angiotensin-converting enzyme inhibitor/angiotensin receptor blocker. RESULTS: In total, 126 cases of SARS-CoV-2 infection were included within a 5-month period, with 16 (13%) and 110 (87%) cases with two and zero/one APOL1 high-risk alleles, respectively. AKI occurred in 11 (69%) patients with two APOL1 high-risk alleles and 39 (35%) patients with zero/one high-risk alleles (adjusted odds ratio, 4.41; 95% confidence interval, 1.11 to 17.52; P=0.04). Persistent AKI occurred in eight (50%) patients with two APOL1 high-risk alleles and 21 (19%) of those with zero/one high-risk alleles (adjusted odds ratio, 3.53; 95% confidence interval, 1.8 to 11.57; P=0.04). AKI KRT occurred in four (25%) of those with two APOL1 high-risk alleles and eight (7%) of those with zero/one high-risk alleles (adjusted odds ratio, 4.99; 95% confidence interval, 1.02 to 24.4, P=0.05). CONCLUSIONS: APOL1 high-risk alleles are associated with greater odds of AKI in Black American patients with COVID-19.

Unbiased Screens Show CD8+ T Cells of COVID-19 Patients Recognize Shared Epitopes in SARS-CoV-2 that Largely Reside outside the Spike Protein.

Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19 patients. In total, we identified 3-8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8+ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8+ T cell immunity to SARS-CoV-2.

Comparison of Biaxial Biomechanical Properties of Post-menopausal Human Prolapsed and Non-prolapsed Uterosacral Ligament.

Uterosacral ligaments (USLs) provide structural support to the female pelvic floor, and a loss of USL structural integrity or biomechanical function may induce pelvic organ prolapse (POP). Alterations in extracellular matrix composition and organization dictate USL mechanical function. Changes in USL microstructure and corresponding mechanical properties, however, are not fully understood, nor is it understood how microstructure and mechanics change with onset and progression of POP. This is due, in part, as USL properties are primarily characterized along a single direction (uniaxial test), whereas the USL is loaded in multiple directions simultaneously within the body. Biaxial testing permits the acquisition of biomechanical data from two axes simultaneously, and thus simulates a more physiologic assessment compared to the traditional uniaxial testing. Therefore, the objective of this study was to quantify the biaxial biomechanical properties and histological composition of the USL in post-menopausal women with and without POP at various stages. Potential correlations between tissue microstructural composition and mechanical function were also examined. Tangential modulus was lower and peak stretch higher in POP III/IV compared to non-POP and POP I/II in the main in vivo loading direction; however, no significant differences in mechanical properties were observed in the perpendicular loading direction. Collagen content positively correlated to tangential modulus in the main in vivo loading direction (r = 0.5, p = 0.02) and negatively correlated with the peak stretch in both the main in vivo (r = -0.5, p = 0.02) and perpendicular loading directions (r = -0.3, p = 0.05). However, no statistically significant differences in USL composition were observed, which may be due to the small sample size and high variability of small sections of human tissues. These results provide first step towards understanding what microstructural and mechanical changes may occur in the USL with POP onset and progression. Such information may provide important future insights into the development of new surgical reconstruction techniques and graft materials for POP treatment.

Cervical and systemic concentrations of long acting hormonal contraceptive (LARC) progestins depend on delivery method: Implications for the study of HIV transmission.

Progestin-only long-acting reversible contraceptives (LARCs) are increasingly popular among women seeking contraception; however, recent epidemiological studies suggest that systemically administered medroxyprogesterone acetate (MPA) may increase HIV acquisition. In order to determine the exact mechanisms underlying increases in transmission specific to MPA use and to test safer, alternative contraceptive progestin types and delivery methods, in vitro modeling studies must be performed. To achieve this, it is imperative that accurate hormone concentrations be utilized when modeling progestin-mediated outcomes, as the down-stream effects are dose-dependent. The local concentrations of progestins to which the lower female genital tract tissues are exposed after initiation of LARCs are unknown, but they likely differ from peripheral concentrations, dependent upon the progestin type and delivery method. Here, we measured in vivo endocervical and plasma concentrations of (1) systemically-delivered depo MPA (DMPA), (2) levonorgestrel (LNG) delivered via intrauterine system (IUS) and (3) etonogestrel (ETG) delivered via vaginal ring in women who recently initiated contraception treatment. Levels of ETG and LNG in cervical secretions were 100-200 fold higher than plasma levels. In contrast, measurable MPA levels were approximately 10-fold higher in plasma compared to cervical secretions. These results will inform the design of accurate in vitro studies on the influence of progestins on epithelial cells, tissue explants, and peripheral blood cells, to be able to better predict in vivo outcomes. Subsequent observations will aid in determining how MPA might influence HIV acquisition and may facilitate identification of optimal progestin-containing LARC alternatives for women at high risk for HIV infection.

A novel whole-bacterial enzyme linked-immunosorbant assay to quantify Chlamydia trachomatis specific antibodies reveals distinct differences between systemic and genital compartments.

Chlamydia trachomatis (CT) is the leading sexually transmitted bacterial infection. The continued global burden of CT infection strongly predicates the need for a vaccine to supplement current chlamydial control programs. The correlates of protection against CT are currently unknown, but they must be carefully defined to guide vaccine design. The localized nature of chlamydial infection in columnar epithelial cells of the genital tract necessitates investigation of immunity at the site of infection. The purpose of this study was to develop a sensitive whole bacterial enzyme-linked immunosorbent assay (ELISA) to quantify and compare CT-specific IgG and IgA in sera and genital secretions from CT-infected women. To achieve this, elementary bodies (EBs) from two of the most common genital serovars (D and E) were attached to poly-L-lysine-coated microtiter plates with glutaraldehyde. EB attachment and integrity were verified by the presence of outer membrane antigens and the absence of bacterial cytoplasmic antigens. EB-specific IgG and IgA standards were developed by pooling sera with high titers of CT-specific antibodies from infected women. Serum, endocervical and vaginal secretions, and endocervical cytobrush specimens from CT-infected women were used to quantify CT-specific IgG and IgA which were then normalized to total IgG and IgA, respectively. Analyses of paired serum and genital samples revealed significantly higher proportions of EB-specific antibodies in genital secretions compared to sera. Cervical and vaginal secretions and cytobrush specimens had similar proportions of EB-specific antibodies, suggesting any one of these genital sampling techniques could be used to quantify CT-specific antibodies when appropriate normalization methodologies are implemented. Overall, these results illustrate the need to investigate genital tract CT antibody responses, and our assay provides a useful quantitative tool to assess natural immunity in defined clinical groups and CT vaccine trials.

Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events.

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIVBaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.

Influence of the tryptophan-indole-IFNγ axis on human genital Chlamydia trachomatis infection: role of vaginal co-infections.

The natural history of genital Chlamydia trachomatis infections can vary widely; infections can spontaneously resolve but can also last from months to years, potentially progressing to cause significant pathology. The host and bacterial factors underlying this wide variation are not completely understood, but emphasize the bacterium's capacity to evade/adapt to the genital immune response, and/or exploit local environmental conditions to survive this immune response. IFNγ is considered to be a primary host protective cytokine against endocervical C. trachomatis infections. IFNγ acts by inducing the host enzyme indoleamine 2,3-dioxgenase, which catabolizes tryptophan, thereby depriving the bacterium of this essential amino acid. In vitro studies have revealed that tryptophan deprivation causes Chlamydia to enter a viable but non-infectious growth pattern that is termed a persistent growth form, characterized by a unique morphology and gene expression pattern. Provision of tryptophan can reactivate the bacterium to the normal developmental cycle. There is a significant difference in the capacity of ocular and genital C. trachomatis serovars to counter tryptophan deprivation. The latter uniquely encode a functional tryptophan synthase to synthesize tryptophan via indole salvage, should indole be available in the infection microenvironment. In vitro studies have confirmed the capacity of indole to mitigate the effects of IFNγ; it has been suggested that a perturbed vaginal microbiome may provide a source of indole in vivo. Consistent with this hypothesis, the microbiome associated with bacterial vaginosis includes species that encode a tryptophanase to produce indole. In this review, we discuss the natural history of genital chlamydial infections, morphological and molecular changes imposed by IFNγ on Chlamydia, and finally, the microenvironmental conditions associated with vaginal co-infections that can ameliorate the effects of IFNγ on C. trachomatis.

Morphologic and molecular evaluation of Chlamydia trachomatis growth in human endocervix reveals distinct growth patterns.

In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence.

Membrane vesicle production by Chlamydia trachomatis as an adaptive response.

Bacteria have evolved specific adaptive responses to cope with changing environments. These adaptations include stress response phenotypes with dynamic modifications of the bacterial cell envelope and generation of membrane vesicles (MVs). The obligate intracellular bacterium, Chlamydia trachomatis, typically has a biphasic lifestyle, but can enter into an altered growth state typified by morphologically aberrant chlamydial forms, termed persistent growth forms, when induced by stress in vitro. How C. trachomatis can adapt to a persistent growth state in host epithelial cells in vivo is not well understood, but is an important question, since it extends the host-bacterial relationship in vitro and has thus been indicated as a survival mechanism in chronic chlamydial infections. Here, we review recent findings on the mechanistic aspects of bacterial adaptation to stress with a focus on how C. trachomatis remodels its envelope, produces MVs, and the potential important consequences of MV production with respect to host-pathogen interactions. Emerging data suggest that the generation of MVs may be an important mechanism for C. trachomatis intracellular survival of stress, and thus may aid in the establishment of a chronic infection in human genital epithelial cells.

Chlamydia trachomatis infection results in a modest pro-inflammatory cytokine response and a decrease in T cell chemokine secretion in human polarized endocervical epithelial cells.

The endocervical epithelium is a major reservoir for Chlamydia trachomatis in women, and genital infections are extended in their duration. Epithelial cells act as mucosal sentinels by secreting cytokines and chemokines in response to pathogen challenge and infection. We therefore determined the signature cytokine and chemokine response of primary-like endocervix-derived epithelial cells in response to a common genital serovar (D) of C. trachomatis. For these studies, we used a recently-established polarized, immortalized, endocervical epithelial cell model (polA2EN) that maintains, in vitro, the architectural and functional characteristics of endocervical epithelial cells in vivo including the production of pro-inflammatory cytokines. PolA2EN cells were susceptible to C. trachomatis infection, and chlamydiae in these cells underwent a normal developmental cycle as determined by a one-step growth curve. IL1α protein levels were increased in both apical and basolateral secretions of C. trachomatis infected polA2EN cells, but this response did not occur until 72h after infection. Furthermore, protein levels of the pro-inflammatory cytokines and chemokines IL6, TNFα and CXCL8 were not significantly different between C. trachomatis infected polA2EN cells and mock infected cells at any time during the chlamydial developmental cycle up to 120h post-infection. Intriguingly, C. trachomatis infection resulted in a significant decrease in the constitutive secretion of T cell chemokines IP10 and RANTES, and this required a productive C. trachomatis infection. Examination of anti-inflammatory cytokines revealed a high constitutive apical secretion of IL1ra from polA2EN cells that was not significantly modulated by C. trachomatis infection. IL-11 was induced by C. trachomatis, although only from the basolateral membrane. These results suggest that C. trachomatis can use evasion strategies to circumvent a robust pro-inflammatory cytokine and chemokine response. These evasion strategies, together with the inherent immune repertoire of endocervical epithelial cells, may aid chlamydiae in establishing, and possibly sustaining, an intracellular niche in microenvironments of the endocervix in vivo.

Potential mechanisms for increased HIV-1 transmission across the endocervical epithelium during C. trachomatis infection.

Among the now pandemic sexually transmitted infections (STIs), Chlamydia trachomatis (C. trachomatis) is the predominant bacterial pathogen and human immunodeficiency virus type 1 (HIV-1) is the most lethal of the viral pathogens. The female genital tract is the primary site for heterosexual transmission of both C. trachomatis and HIV-1. Infection with C. trachomatis, and with a variety of other STIs, increases the risk for transmission of HIV-1, although the mechanisms for this finding remain unclear. We have used in vitro modeling to assess the mechanisms by which infection with genital C. trachomatis serovars might increase the transmission of HIV-1 across the female genital tract. C. trachomatis infection of an immortalized endocervical epithelial cell line (A2EN) increases the cell surface expression of the HIV-1 alternative primary receptor, galactosyl ceramide (GalCer), and of the HIV-1 co-receptors, CXCR4 and CCR5. C. trachomatis infection also increases the binding of HIV-1 to A2EN cells, and, subsequently, increases levels of virus in co-cultures of HIV-exposed A2EN and susceptible MT4-R5 T cells. Finally, in vivo endocervical cell sampling reveals a dramatic increase in the number of CD4+, CXCR4 and/or CCR5 positive T cell targets in the endocervix of C. trachomatis positive women when compared to those who are C. trachomatis negative. This combination of in vitro and in vivo results suggests several mechanisms for increased transmission of HIV-1 across the endocervices of C. trachomatis-infected women.

Innate immune mediator profiles and their regulation in a novel polarized immortalized epithelial cell model derived from human endocervix.

The endocervix in the female reproductive tract (FRT) is susceptible to sexually transmitted pathogens such as Chlamydia trachomatis and Neisseria gonorrhoeae. Endocervical epithelial cells in vivo make innate immune mediators that likely aid in the protection from these pathogens. In vitro studies to investigate the innate epithelial cell immune response to endocervical pathogens have been hindered by the paucity of human endocervix-derived epithelial cell lines that display the differentiation proteins and functional characteristics of their site of origin. We have established an immortalized epithelial cell line (A2EN) derived from an endocervical tissue explant that can be polarized to exhibit distinct apical and basolateral membrane domains. Polarized A2EN cells secrete mucus at their apical surface, and express MUC5B, a mucin specific to the endocervix. Polarized A2EN cells also express hormone receptors that respond appropriately to female steroid hormones. Polarized A2EN cells can be stimulated with the toll-like receptor 3 agonist, polyI:C, to express anti-microbial peptides (AMPs) as well as pro-inflammatory cytokines and chemokines. Cytokines and chemokines are also differentially secreted depending on the hormone milieu in which the cells are exposed. We conclude that polarized A2EN cells maintain distinctive phenotypic and functional characteristics of the epithelial cells found in the endocervix and, hence, could provide a useful, new in vitro model system for investigations on the role of endogenous and exogenous factors that regulate endocervical epithelial cell immunity including studies on sexually transmitted infections and topical microbicides.

Altered protein secretion of Chlamydia trachomatis in persistently infected human endocervical epithelial cells.

Chlamydia trachomatis is the most common bacterial infection of the human reproductive tract globally; however, the mechanisms underlying the adaptation of the organism to its natural target cells, human endocervical epithelial cells, are not clearly understood. To secure its intracellular niche, C. trachomatis must modulate the host cellular machinery by secreting virulence factors into the host cytosol to facilitate bacterial growth and survival. Here we used primary human endocervical epithelial cells and HeLa cells infected with C. trachomatis to examine the secretion of bacterial proteins during productive growth and persistent growth induced by ampicillin. Specifically, we observed a decrease in secretable chlamydial protease-like activity factor (CPAF) in the cytosol of host epithelial cells exposed to ampicillin with no evident reduction of CPAF product by C. trachomatis. In contrast, the expression of CopN and Tarp was downregulated, suggesting that C. trachomatis responds to ampicillin exposure by selectively altering the expression of secretable proteins. In addition, we observed a greater accumulation of outer-membrane vesicles from C. trachomatis in persistently infected cells. Taken together, these results suggest that the regulation of both gene expression and the secretion of chlamydial virulence proteins is involved in the adaptation of the bacteria to a persistent infection state in human genital epithelial cells.