Racial Differences in Plasma Microbial Translocation and Plasma Microbiome, Implications in Systemic Lupus Erythematosus Disease Pathogenesis.

OBJECTIVE: Black groups have increased prevalence and accelerated pathogenicity of systemic lupus erythematosus (SLE) compared to other ethnic/racial groups. The microbiome and systemic microbial translocation are considered contributing factors to SLE disease pathogenesis. However, racial differences in the plasma microbiome and microbial translocation in lupus remain unknown. METHODS: In the current study, we investigated plasma levels of microbial translocation (lipopolysaccharide [LPS] and zonulin) and the plasma microbiome using microbial 16S RNA sequencing of Black and White patients with SLE and Black and White healthy controls. RESULTS: Plasma microbial translocation was increased in Black patients versus in White patients and in patients with SLE versus healthy controls regardless of race. Compared to sex, age, and disease status, race had the strongest association with plasma microbiome differences. Black groups (Black controls and Black patients) had lower α-diversity than White groups (White controls and White patients) and more distinct ß-diversity. Black and White patients demonstrated differences in plasma bacterial presence, including Staphylococcus and Burkholderia. Compared to White patients, Black patients had higher SLE Disease Activity Index (SLEDAI) scores and urinary protein levels as well as a trend for increased anti-double-stranded DNA (dsDNA) antibody levels consistent with the known increased severity of lupus in Black patients overall. Certain plasma bacteria at the genus level were identified that were associated with the SLEDAI score, urinary protein, and anti-dsDNA antibody levels. CONCLUSION: This study reveals racial differences in both quality and quantity of plasma microbial translocation and identified specific plasma microbiome differences associated with SLE disease pathogenesis. Thus, this study may provide new insights into future potential microbiome therapies on SLE pathogenesis.

Staphylococcus aureus peptidoglycan (PGN) induces pathogenic autoantibody production via autoreactive B cell receptor clonal selection, implications in systemic lupus erythematosus.

OBJECTIVES: There is an intricate interplay between the microbiome and the immune response impacting development of normal immunity and autoimmunity. However, we do not fully understand how the microbiome affects production of natural-like and pathogenic autoantibodies. Peptidoglycan (PGN) is a component of the bacterial cell wall which is highly antigenic. PGNs from different bacteria can differ in their immune regulatory activities. METHODS: C57BL/6 and MRL/lpr mice were intraperitoneally injected with saline or PGN from Staphylococcus aureus or Bacillus subtilis. Spleen anti-double-stranded DNA (dsDNA) IgG + B cells were sorted for B-cell receptor sequencing. Serum autoantibody levels and kidney damage were analyzed. Further, the association between plasma S. aureus translocation and systemic lupus erythematosus (SLE) pathogenesis was assessed in women. RESULTS: Administration of B. subtilis PGN induced natural-like anti-dsDNA autoantibodies (e.g., IgM, short lived IgG response, and no tissue damage), whereas S. aureus PGN induced pathogenic anti-dsDNA autoantibodies (e.g., prolonged IgG production, low IgM, autoantibody-mediated kidney damage) in C57BL/6 and/or MRL/lpr mice. However, serum total IgG did not differ. S. aureus PGN induced antibodies with reduced clonality and greater hypermutation of IGHV3-74 in splenic anti-dsDNA IgG + B cells from C57BL/6 mice. Further, S. aureus PGN promoted IgG class switch recombination via toll-like receptor 2. Plasma S. aureus DNA levels were increased in women with SLE versus control women and correlated with levels of lupus-related autoantibodies and renal involvement. CONCLUSIONS: S. aureus PGN induces pathogenic autoantibody production, whereas B. subtilis PGN drives production of natural nonpathogenic autoantibodies.

A Distinct Plasma Microbiome But Not Gut Microbiome in Patients With Systemic Lupus Erythematosus Compared to Healthy Individuals.

OBJECTIVE: Blood microbiome has been analyzed in cancer patients using machine learning. We aimed to study whether the plasma microbiome represents the microbial community in the gut among patients with systemic lupus erythematosus (SLE) and healthy controls (HCs). METHODS: Paired plasma and stool samples from female patients with SLE and female HCs were assessed for microbiome composition by microbial 16S ribosomal RNA sequencing. RESULTS: Decreased microbial alpha diversity in stool compared to plasma and distinct plasma and gut beta diversity were found in both HCs and patients with SLE. No difference in gut microbial diversity was found; however, plasma alpha diversity was decreased in patients with SLE compared to HCs. The predominant bacteria differed between plasma and stool in both groups. Although the predominant plasma and stool genus bacteria were similar in patients with SLE and HCs, some were clearly different. CONCLUSION: Compared to the gut, the plasma microbiome contained distinct community and greater heterogeneity, indicating that the predominant circulating microbiome may originate from sites (eg, oral or skin) other than the gastrointestinal tract. The decreased plasma but not gut alpha diversity in patients with SLE compared to HCs implies an altered plasma microbiome in SLE, which may be important for systemic immune perturbations and SLE disease pathogenesis.

Rigorous Plasma Microbiome Analysis Method Enables Disease Association Discovery in Clinic.

Blood microbiome is important to investigate microbial-host interactions and the effects on systemic immune perturbations. However, this effort has met with major challenges due to low microbial biomass and background artifacts. In the current study, microbial 16S DNA sequencing was applied to analyze plasma microbiome. We have developed a quality-filtering strategy to evaluate and exclude low levels of microbial sequences, potential contaminations, and artifacts from plasma microbial 16S DNA sequencing analyses. Furthermore, we have applied our technique in three cohorts, including tobacco-smokers, HIV-infected individuals, and individuals with systemic lupus erythematosus (SLE), as well as corresponding controls. More than 97% of total sequence data was removed using stringent quality-filtering strategy analyses; those removed amplicon sequence variants (ASVs) were low levels of microbial sequences, contaminations, and artifacts. The specifically enriched pathobiont bacterial ASVs have been identified in plasmas from tobacco-smokers, HIV-infected individuals, and individuals with SLE but not from control subjects. The associations between these ASVs and disease pathogenesis were demonstrated. The pathologic activities of some identified bacteria were further verified in vitro. We present a quality-filtering strategy to identify pathogenesis-associated plasma microbiome. Our approach provides a method for studying the diagnosis of subclinical microbial infection as well as for understanding the roles of microbiome-host interaction in disease pathogenesis.

Progesterone decreases gut permeability through upregulating occludin expression in primary human gut tissues and Caco-2 cells.

Progesterone plays a protective role in preventing inflammation and preterm delivery during pregnancy. However, the mechanism involved is unknown. Microbial product translocation from a permeable mucosa is demonstrated as a driver of inflammation. To study the mechanism of the protective role of progesterone during pregnancy, we investigated the effect of physiologic concentrations of progesterone on tight junction protein occludin expression and human gut permeability in vitro and systemic microbial translocation in pregnant women in vivo. Plasma bacterial lipopolysaccharide (LPS), a representative marker of in vivo systemic microbial translocation was measured. We found that plasma LPS levels were significantly decreased during 24 to 28 weeks of gestation compared to 8 to 12 weeks of gestation. Moreover, plasma LPS levels were negatively correlated with plasma progesterone levels but positively correlated with plasma tumor necrosis factor-alpha (TNF-α) levels at 8 to 12 weeks of gestation but not at 24 to 28 weeks of gestation. Progesterone treatment increased intestinal trans-epithelial electrical resistance (TEER) in primary human colon tissues and Caco-2 cells in vitro through upregulating tight junction protein occludin expression. Furthermore, progesterone exhibited an inhibitory effect on nuclear factor kappa B (NF-κB) activation following LPS stimulation in Caco-2 cells. These results reveal a novel mechanism that progesterone may play an important role in decreasing mucosal permeability, systemic microbial translocation, and inflammation during pregnancy.

A Link Between Plasma Microbial Translocation, Microbiome, and Autoantibody Development in First-Degree Relatives of Systemic Lupus Erythematosus Patients.

OBJECTIVE: Systemic lupus erythematosus (SLE) is characterized by the production of antibodies against self antigens. However, the events underlying autoantibody formation in SLE remain unclear. This study was undertaken to investigate the role of plasma autoantibody levels, microbial translocation, and the microbiome in SLE. METHODS: Plasma samples from 2 cohorts, one with 18 unrelated healthy controls and 18 first-degree relatives and the other with 19 healthy controls and 21 SLE patients, were assessed for autoantibody levels by autoantigen microarray analysis, measurement of lipopolysaccharide (LPS) levels by Limulus amebocyte assay, and determination of microbiome composition by microbial 16S ribosomal DNA sequencing. RESULTS: First-degree relatives and SLE patients exhibited increased plasma autoantibody levels compared to their control groups. Parents and children of lupus patients exhibited elevated plasma LPS levels compared to controls (P = 0.02). Plasma LPS levels positively correlated with plasma anti-double-stranded DNA IgG levels in first-degree relatives (r = 0.51, P = 0.03), but not in SLE patients. Circulating microbiome analysis revealed that first-degree relatives had significantly reduced microbiome diversity compared to their controls (observed species, P = 0.004; Chao1 index, P = 0.005), but this reduction was not observed in SLE patients. The majority of bacteria that were differentially abundant between unrelated healthy controls and first-degree relatives were in the Firmicutes phylum, while differences in bacteria from several phyla were identified between healthy controls and SLE patients. Bacteria in the Paenibacillus genus were the only overlapping differentially abundant bacteria in both cohorts, and were reduced in first-degree relatives (adjusted P [Padj ] = 2.13 × 10-12 ) and SLE patients (Padj = 0.008) but elevated in controls. CONCLUSIONS: These results indicate a possible role of plasma microbial translocation and microbiome composition in influencing autoantibody development in SLE.

Systemic translocation of Staphylococcus drives autoantibody production in HIV disease.

BACKGROUND: Increased autoreactive antibodies have been reported in HIV disease; however, the mechanism accounting for autoantibody induction in HIV remains unknown. RESULTS: Herein, we show that seasonal influenza vaccination induces autoantibody production (e.g., IgG anti-nuclear antibody (ANA) and anti-double-stranded DNA antibody (anti-dsDNA)) in some viral-suppressed antiretroviral therapy (ART)-treated HIV+ subjects, but not in healthy controls. These autoantibodies were not derived from antigen-specific B cells but from activated "bystander" B cells analyzed by single-cell assay and by study of purified polyclonal ANAs from plasma. To explore the mechanism of autoantibody generation in HIV+ subjects, plasma level of microbial products, gene expression profile of B cells, and B cell receptor (BCR) repertoires were analyzed. We found that autoantibody production was associated with increased plasma level of microbial translocation; the patients with high autoantibodies had skewed B cell repertoires and upregulation of genes related to innate immune activation in response to microbial translocation. By analyzing circulating microbial 16S rDNA in plasma, the relative abundance of Staphylococcus was found to be associated with autoantibody production in HIV+ subjects. Finally, we found that injection of heat-killed Staphylococcus aureus promoted germinal center B cell responses and autoantibody production in mice, consistent with the notion that autoantibody production in HIV+ patients is triggered by microbial products. CONCLUSIONS: Our results showed that translocation of Staphylococcus can promote B cell activation through enhancing germinal center response and induces autoantibody production. It uncovers a potential mechanism linking microbial translocation and autoimmunity in HIV+ disease and provides a strong rationale for targeting Staphylococcus to prevent autoantibody production.

Increased influenza-specific antibody avidity in HIV-infected women compared with HIV-infected men on antiretroviral therapy.

BACKGROUND: It is recommended that HIV-infected individuals receive annual influenza vaccination due to their high susceptibility to influenza infection, especially among women. However, there have been few studies investigating sex-related responses to influenza vaccine in antiretroviral therapy (ART)-treated HIV-infected individuals. METHOD: In this study, 26 aviremic ART-treated HIV-infected individuals and 16 healthy controls were enrolled in the current study. Blood was collected prior to vaccination (D0), on days 7-10 (D7) and on days 14-21 (D14) following administration of the 2013-2014 seasonal influenza vaccine. A series of analyses evaluated the serological and cellular responses following influenza vaccination. RESULTS: Female HIV-infected individuals had increased influenza-specific antibody avidity relative to male HIV-infected individuals, but similar plasma levels of influenza-specific binding antibodies and neutralizing antibodies. Increased cycling B cells and follicular helper CD4 T (Tfh) cells were observed in female HIV-infected individuals pre and postvaccination compared with male HIV-infected individuals, and cycling Tfh cells were directly correlated with influenza-specific antibody avidity. Moreover, plasma testosterone levels were inversely correlated with antibody avidity index. The magnitude of microbial translocation [plasma lipopolysaccharide (LPS)] level was directly correlated with influenza-specific antibody avidity. Circulating 16S rDNA microbiome showed that enrichment of specific species within Proteobacteria was associated with influenza-specific antibody avidity. These results, including differences based on sex and correlations, were only observed in HIV-infected individuals but not in the healthy controls. CONCLUSION: This study demonstrated sex differences in influenza-specific antibody avidity in ART-treated HIV disease, and provides valuable information on vaccination strategy in the ART-treated HIV-infected population.

Increased systemic microbial translocation is associated with depression during early pregnancy.

Plasma level of microbial translocation is a marker of mucosal permeability. Increased mucosal permeability ignites elevated microbial translocation and as a consequence of systemic inflammation. Pregnant women with depression have higher levels of inflammatory markers relative to pregnant women without depression, however, no studies have reported whether systemic microbial translocation will change in depressed women during pregnancy. In this study, we examined the plasma LPS level of depressed women during pregnancy. The results showed that the plasma LPS level was significantly increased in depressed mothers during their 8-12 weeks gestation compared to healthy controls. Compared to 8-12 weeks gestation, the plasma LPS levels were significantly decreased at 24-28 weeks gestation and 6-8 weeks postpartum in both depressed subjects and healthy controls. Furthermore, the plasma levels of pro-inflammatory cytokines (TNF-α and MCP/CCL2) associated with microbial translocation were significantly increased in depressed subjects during 8-12 weeks gestation compared to healthy controls. These results indicate that the level of microbial translocation is increased in depressed women during early pregnancy.

The effect of plasma auto-IgGs on CD4+ T cell apoptosis and recovery in HIV-infected patients under antiretroviral therapy.

Although effective antiretroviral therapy (ART) suppresses HIV viral replication, prevents AIDS-related complications, and prolongs life, a proportion of patients fails to restore the patients' CD4+ T cell number to the level of healthy individuals. Increased mortality and morbidity have been observed in these patients. In the current study, we have investigated the role of auto-IgGs in CD4+ T cell apoptosis and recovery in a cross-sectional study. All HIV+ subjects were on viral-suppressive ART treatment with a different degree of CD4+ T cell reconstitution. Total auto-IgG binding on CD4+ T cell surfaces and its associated apoptosis and CD4+ T cell recovery were analyzed by flow cytometry ex vivo. Total IgGs from plasma were tested for their binding capacities to CD4+ T cell surfaces and their mediation to CD4+ T cell death through NK cell cytotoxicity in vitro. HIV+ subjects had increased surface binding of auto-IgGs on CD4+ T cells compared with healthy controls, and IgG binding was associated with elevated CD4+ T cell apoptosis in HIV+ subjects but not in healthy controls. Plasma IgGs from HIV+ subjects bound to CD4+ T cells and induced cell apoptosis through NK cytotoxicity in vitro. Soluble CD4 (sCD4) preincubation prevented NK cell-mediated CD4+ T cell death. Our results suggest that plasma autoantibodies may play a role in some HIV+ patients with poor CD4+ T cell recovery under viral-suppressive ART.