Autocrine stimulation of IL-10 is critical to the enrichment of IL-10-producing CD40hiCD5+ regulatory B cells in vitro and in vivo

IL-10-producing B (Breg) cells regulate various immune responses. However, their phenotype remains unclear. CD40 expression was significantly increased in B cells by LPS, and the Breg cells were also enriched in CD40^hiCD5⁺ B cells. Furthermore, CD40 expression on Breg cells was increased by IL-10, CD40 ligand, and B cell-activating factor, suggesting that CD40^hi is a common phenotype of Breg cells. LPS-induced CD40 expression was largely suppressed by an anti-IL-10 receptor antibody and in IL-10^−/−CD5⁺CD19⁺ B cells. The autocrine effect of IL-10 on the CD40 expression was largely suppressed by an inhibitor of JAK/STAT3. In vivo, the LPS treatment increased the population of CD40^hiCD5⁺ Breg cells in mice. However, the population of CD40^hiCD5⁺ B cells was minimal in IL-10^−/− mice by LPS. Altogether, our findings show that Breg cells are largely enriched in CD40^hiCD5⁺ B cells and the autocrine effect of IL-10 is critical to the formation of CD40^hiCD5⁺ Breg cells. [BMB Reports 2015; 48(1): 54-59]     

An age‐related numerical and functional deficit in CD19+CD24hiCD38hi B cells is associated with an increase in systemic autoimmunity

Autoimmunity increases with aging indicative of reduced immune tolerance, but the mechanisms involved are poorly defined. In recent years, subsets of B cells with immunoregulatory properties have been identified in murine models of autoimmune disorders, and these cells downregulate immune responses via secretion of IL10. In humans, immature transitional B cells with a CD19⁺CD24^hiCD38^hi phenotype have been reported to regulate immune responses via IL10 production. We found the frequency and numbers of CD19⁺CD24^hiCD38^hi cells were reduced in the PBMC pool with age. IL10 expression and secretion following activation via either CD40, or Toll‐like receptors was also impaired in CD19⁺CD24^hiCD38^hi B cells from healthy older donors. When investigating the mechanisms involved, we found that CD19⁺CD24^hiCD38^hi B‐cell function was compromised by age‐related effects on both T cells and B cells: specifically, CD40 ligand expression was lower in CD4 T cells from older donors following CD3 stimulation, and signalling through CD40 was impaired in CD19⁺CD24^hiCD38^hi B cells from elders as evidenced by reduced phosphorylation (Y705) and activation of STAT3. However, there was no age‐associated change in expression of costimulatory molecules CD80 and CD86 on CD19⁺CD24^hiCD38^hi cells, suggesting IL10‐dependent immune suppression is impaired, but contact‐dependent suppressive capacity is intact with age. Finally, we found a negative correlation between CD19⁺CD24^hiCD38^hi B‐cell IL10 production and autoantibody (Rheumatoid factor) levels in older adults. We therefore propose that an age‐related decline in CD19⁺CD24^hiCD38^hi B cell number and function may contribute towards the increased autoimmunity and reduced immune tolerance seen with aging.     

Orally-Induced Intestinal CD4+ CD25+ FoxP3+ Treg Controlled Undesired Responses towards Oral Antigens and Effectively Dampened Food Allergic Reactions

The induction of peripheral tolerance may constitute a disease-modifying treatment for allergic patients. We studied how oral immunotherapy (OIT) with milk proteins controlled allergy in sensitized mice (cholera toxin plus milk proteins) upon exposure to the allergen. Symptoms were alleviated, skin test was negativized, serum specific IgE and IgG1 were abrogated, a substantial reduction in the secretion of IL-5 and IL-13 by antigen-stimulated spleen cells was observed, while IL-13 gene expression in jejunum was down-regulated, and IL-10 and TGF-β were increased. In addition, we observed an induction of CD4⁺CD25⁺FoxP3⁺ cells and IL-10- and TGF-β-producing regulatory T cells in the lamina propria. Finally, transfer experiments confirmed the central role of these cells in tolerance induction. We demonstrated that the oral administration of milk proteins pre- or post-sensitization controlled the Th2-immune response through the elicitation of mucosal IL-10- and TGF-β-producing Tregs that inhibited hypersensitivity symptoms and the allergic response.     

Interleukin-5 Supports the Expansion of Fas Ligand-Expressing Killer B Cells that Induce Antigen-Specific Apoptosis of CD4+ T Cells and Secrete Interleukin-10

Beyond their critical role in humoral immunity, B lymphocytes can employ a variety of immunomodulatory mechanisms including expression of the apoptosis-inducing molecule Fas ligand (FasL; CD178). Here, we extensively characterized the surface phenotype of FasL⁺ killer B cells, showing they are enriched in the IgM^highCD5⁺CD1d^high B cell subset previously reported to contain a higher frequency of B cells producing interleukin-10 (IL-10). A rare population of B cells expressing IL-10 was present among FasL⁺ B cells, but most FasL⁺ B cells did not produce IL-10. We also identify interleukin-5 (IL-5) as a novel inducer of killer B cell function. Constitutively FasL⁺ B cells expressed higher levels of the IL-5 receptor, and treating B cells with IL-5 and CD40L resulted in the expansion of a B cell population enriched for FasL⁺ cells. B cells stimulated with IL-5 and CD40L were potent inducers of apoptosis in activated primary CD4⁺ T cells, and this killing function was antigen-specific and dependent upon FasL. IL-5 also enhanced IL-10 secretion in B cells stimulated with CD40L. Taken together these findings elucidate the relationship of FasL⁺ B cells and IL-10-producing B cells and demonstrate that IL-5 can induce or enhance both killer B cell activity and IL-10 secretion in B cells. Finally, we found that the killer B cell activity induced by IL-5 was completely blocked by IL-4, suggesting the existence of a previously unknown antagonistic relationship between these type-2 cytokines in modulating the activity of killer B cells. Targeting this IL-5/IL-4 signaling axis may therefore represent a novel area of drug discovery in inflammatory disorders.     

IL-10-Producing Regulatory B Cells Are Decreased in Patients with Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency in adults. CVID patients often present changes in the frequency and function of B lymphocytes, reduced number of Treg cells, chronic immune activation, recurrent infections, high incidence of autoimmunity and increased risk for malignancies. We hypothesized that the frequency of B10 cells would be diminished in CVID patients because these cells play an important role in the development of Treg cells and in the control of T cell activation and autoimmunity. Therefore, we evaluated the frequency of B10 cells in CVID patients and correlated it with different clinical and immunological characteristics of this disease. Forty-two CVID patients and 17 healthy controls were recruited for this study. Cryopreserved PBMCs were used for analysis of T cell activation, frequency of Treg cells and characterization of B10 cells by flow cytometry. IL-10 production by sorted B cells culture and plasma sCD14 were determined by ELISA. We found that CVID patients presented decreased frequency of IL-10-producing CD24^hiCD38^hi B cells in different cell culture conditions and decreased frequency of IL-10-producing CD24^hiCD27⁺ B cells stimulated with CpG+PIB. Moreover, we found that CVID patients presented lower secretion of IL-10 by sorting-purified B cells when compared to healthy controls. The frequency of B10 cells had no correlation with autoimmunity, immune activation and Treg cells in CVID patients. This work suggests that CVID patients have a compromised regulatory B cell compartment which is not correlated with clinical and immunological characteristics presented by these individuals.     

Peripheral regulatory cells immunophenotyping in Primary Sj?gren's Syndrome: a cross-sectional study

Introduction

IL-10--producing B cells, Foxp3-expressing T cells (Tregs) and the IDO-expressing dendritic cells (pDC) are able to modulate inflammatory processes, to induce immunological tolerance and, in turn, to inhibit the pathogenesis of autoimmune disease.The aim of the study was to characterize and to enumerate peripheral IL-10--producing B cells, Tregs and pDCregs in primary Sjögren''s Syndrome (pSS) patients in regard of their clinical and serologic activity.

Methods

Fifty pSS patients and 25 healthy individuals were included in the study. CD19⁺--expressing peripheral B lymphocytes were purified by positive selection. CD19⁺/CD24^hi/CD38^hi/IL-10--producing B cells, CD4⁺/CD25^hi/Foxp3⁺ and CD8⁺/CD28^-/Foxp3⁺ Tregs, as well as CCR6⁺/CD123⁺/IDO⁺ DCs, were quantitated by flow cytometry.

Results

Immature/transitional circulating IgA⁺ IL-10--producing B cells had higher levels in pSS patients versus control group, whereas CD19⁺/CD38^hi/IgG⁺/IL-10⁺ cells had lower percentage versus control. Indeed CD19⁺/CD24^hi/CD38^hi/CD5⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD10⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD20⁺/IL-10⁺, CD19⁺/CD24^hi/CD38^hi/CD27^-/IL-10⁺, and CD19⁺/CD24^hi/CD38^hi/CXCR7⁺/IL-10⁺ cells had higher frequency in clinical inactive pSS patients when compared with control group. Remarkably, only percentages of CD19⁺/CD24^hi/CD38^hi/CD10⁺/IL-10⁺ and CD19⁺/CD24^hi/CD38^hi/CD27^-/IL-10⁺ subsets were increased in pSS serologic inactive versus control group (P < 0.05). The percentage of IDO-expressing pDC cells was higher in pSS patients regardless of their clinical or serologic activity. There were no statistically significant differences in the percentage of CD4⁺/CD25^hi/Foxp3⁺ Tregs between patient groups versus controls. Nonetheless, a decrease in the frequency of CD8⁺/CD28^-/Foxp3⁺ Tregs was found in inactive pSS patients versus controls (P < 0.05).

Conclusions

The findings of this exploratory study show that clinical inactive pSS patients have an increased frequency of IL-10--producing B cells and IDO-expressing pDC cells.     

Induction of IL-10-Producing CD1dhighCD5+ Regulatory B Cells following Babesia microti-Infection

Background

Understanding the induction of immune regulatory cells upon helminth infection is important for understanding the control of autoimmunity and allergic inflammation in helminth infection. Babesia microti, an intraerythrocytic protozoan of the genus Babesia, is a major cause of the emerging human disease babesiosis, an asymptomatic malaria-like disease. We examined the influence of acute B. microti infection on the development of regulatory B cells together with regulatory T cells.

Principal Findings

Our data demonstrate that B cells stimulated in vitro with B. microti produce interleukin (IL)-10. This cytokine is also secreted by B cells isolated from B. microti-infected mice in response to lipopolysaccharide stimulation. In addition, high levels of IL-10 were detected in the serum of mice after infection with B. microti. The frequency of IL-10-producing CD1d^highCD5⁺ regulatory B cells (Bregs) and CD4⁺CD25⁺FoxP3⁺ T cells increased during the course of B. microti infection. Furthermore, adoptive transfer of IL-10-producing B cells induced by B. microti infection led to increased susceptibility of recipient mice to infection with B. microti. In contrast, experiments with B cell-deficient (µMT) mice demonstrated that lack of B cells enhances susceptibility to B. microti infection.

Conclusions

This study is the first demonstration of the expansion of Bregs following infection by an intraerythrocytic protozoan parasite. These data suggest that B. microti infection in mice provides an excellent model for studying Breg-mediated immune responses and begins to elucidate the mechanism by which helminth infection regulates autoimmunity and allergic inflammation.     

A Review on Biology and Function of Regulatory B Cells with Special Reference to Inflammation and Autoimmune Pathogenesis

Once considered to be predominantly a positive regulator of immune function, B cells are increasingly getting attributed with new and diversified functions. In recent years a number of reports indicate that a definite regulatory function of B cell exists. A number of B cell subsets that shares various developmental markers shown to have the ability to produce the suppressive cytokine IL-10. A particularly strong candidate with distinct IL-10 production profile is CD19^hiCD1d^hiCD5⁺ population, which constitutes about 1–2 % of splenic B cells. These B cells are also called B10 cells as they produce large amount of IL-10. Increasingly it is becoming clear that a variety of regulatory B cell population exists and expands during inflammatory condition and autoimmune disease. A detailed study of development and mechanism that governs B reg generation may provide us with valuable information for therapeutic intervention.     

TRAF3 Regulates Homeostasis of CD8+ Central Memory T Cells

Our laboratory reported previously that TNF receptor associated factor 3 (TRAF3) is a positive regulator of TCR signaling and T cell function. In the current study, we present new findings that reveal differential roles for TRAF3 in the regulation of CD4⁺ and CD8⁺ T cells. In response to TCR stimulation in vitro, TRAF3 has greater impact in CD4⁺ T cells than in CD8⁺ T cells. However, T cell-specific TRAF3 deficient mice (CD4^Cre TRAF3^fl°^x/fl°^x; T-TRAF3^−/−) have a greater number of CD4⁺CD44^hi effector/memory T cells than littermate control (LMC) mice, possibly due to an inefficient suppressive effect of TRAF3 deficient Foxp3⁺ regulatory T cells. In contrast, CD8⁺CD44^hiCD62L^hi central memory (Tcm) cells are markedly reduced in T-TRAF3^−/− mice in comparison to LMC mice, although CD8⁺CD44^hiCD62L^l°^w effector memory T (Tem) cells and naïve T cells (CD8⁺CD44^l°^wCD62L^hi) do not show significant differences in number. Importantly, TRAF3-deficient Tcm cells exhibit defective homeostasis due to impaired IL-15 signaling. These results indicate that the involvement of TRAF3 in IL-15 mediated signaling to T cells plays a previously unappreciated and critical role in CD8⁺ Tcm cell regulation and maintenance.     

Age-Dependent Defects of Regulatory B Cells in Wiskott-Aldrich Syndrome Gene Knockout Mice

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19⁺CD1d^highCD5⁺ B cells (CD1d^highCD5⁺ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1d^highCD5⁺ Breg cells as compared to age-matched WT control mice. CD1d^highCD5⁺ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4⁺ T cells. Interestingly, CD1d^highCD5⁺ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.     

CD4+CD25hiCD127low Regulatory T Cells Are Increased in Oral Squamous Cell Carcinoma Patients

Regulatory T cells (Tregs), a subset of CD4⁺ T cells plays a pivotal role in regulating the immune system. An increase in Treg numbers enables cancer progression by dampening the immune system and allowing tumor cells to evade immune detection and destruction. An increase in Treg numbers and expression of inhibitory cytokines including TGF-β and IL-10 are mechanisms by which Tregs exert their immune suppressive function. However, the presence of Tregs and inhibitory cytokines in oral cancer patients is still unclear. In this study, the presence of circulating Tregs in 39 oral cancer patients and 24 healthy donors was examined by studying the presence of the CD4⁺CD25^hiCD127^low cell population in their peripheral blood mononuclear cells using flow cytometry. Serum levels of TGF-β and IL-10 were measured by ELISA. T cell subsets of OSCC patients were found to differ significantly from healthy donors where a decrease in CD8⁺ cytotoxic T cells and an increase in Tregs (CD4⁺CD25^hiCD127^low) were observed. Further, the ratio of CD8⁺ T cells/Tregs was also decreased in patients compared to healthy donors. The presence of Tregs was accompanied by a decrease in IL-10 but not TGF-β secretion in OSCC patients when compared to donors; in addition, the analysis also revealed that an increased presence of Tregs was accompanied by better patient survival. Amongst OSCC patients, smokers had significantly higher levels of TGF-β. It is apparent that the immune system is compromised in OSCC patients and the characterization of the Treg subpopulation could form a basis for improving our understanding of the perturbations in the immune system that occur during OSCC tumorigenesis.     

TLR5 Signaling Enhances the Proliferation of Human Allogeneic CD40-Activated B Cell Induced CD4hiCD25+ Regulatory T Cells

Although diverse functions of different toll-like receptors (TLR) on human natural regulatory T cells have been demonstrated recently, the role of TLR-related signals on human induced regulatory T cells remain elusive. Previously our group developed an ex vivo high-efficient system in generating human alloantigen-specific CD4^hiCD25⁺ regulatory T cells from naïve CD4⁺CD25⁻ T cells using allogeneic CD40-activated B cells as stimulators. In this study, we investigated the role of TLR5-related signals on the generation and function of these novel CD4^hiCD25⁺ regulatory T cells. It was found that induced CD4^hiCD25⁺ regulatory T cells expressed an up-regulated level of TLR5 compared to their precursors. The blockade of TLR5 using anti-TLR5 antibodies during the co-culture decreased CD4^hiCD25⁺ regulatory T cells proliferation by induction of S phase arrest. The S phase arrest was associated with reduced ERK1/2 phosphorylation. However, TLR5 blockade did not decrease the CTLA-4, GITR and FOXP3 expressions, and the suppressive function of CD4^hiCD25⁺ regulatory T cells. In conclusion, we discovered a novel function of TLR5-related signaling in enhancing the proliferation of CD4^hiCD25⁺ regulatory T cells by promoting S phase progress but not involved in the suppressive function of human CD40-activated B cell-induced CD4^hiCD25⁺ regulatory T cells, suggesting a novel role of TLR5-related signals in the generation of induced regulatory T cells.     

IL-10-Producing Th1 Cells and Disease Progression Are Regulated by Distinct CD11c+ Cell Populations during Visceral Leishmaniasis

IL-10 is a critical regulatory cytokine involved in the pathogenesis of visceral leishmaniasis caused by Leishmania donovani and clinical and experimental data indicate that disease progression is associated with expanded numbers of CD4⁺ IFNγ⁺ T cells committed to IL-10 production. Here, combining conditional cell-specific depletion with adoptive transfer, we demonstrate that only conventional CD11c^hi DCs that produce both IL-10 and IL-27 are capable of inducing IL-10-producing Th1 cells in vivo. In contrast, CD11c^hi as well as CD11c^int/lo cells isolated from infected mice were capable of reversing the host protective effect of diphtheria toxin-mediated CD11c⁺ cell depletion. This was reflected by increased splenomegaly, inhibition of NO production and increased parasite burden. Thus during chronic infection, multiple CD11c⁺ cell populations can actively suppress host resistance and enhance immunopathology, through mechanisms that do not necessarily involve IL-10-producing Th1 cells.     

ICOS Regulates the Generation and Function of Human CD4+ Treg in a CTLA-4 Dependent Manner

Inducible co-stimulator (ICOS) is a member of CD28/Cytotoxic T-lymphocyte Antigen-4 (CTLA-4) family and broadly expressed in activated CD4⁺ T cells and induced regulatory CD4⁺ T cells (CD4⁺ iTreg). ICOS-related signal pathway could be activated by the interaction between ICOS and its ligand (ICOSL). In our previous work, we established a cost-effective system to generate a novel human allo-antigen specific CD4^hi Treg by co-culturing their naïve precursors with allogeneic CD40-activated B cells in vitro. Here we investigate the role of ICOS in the generation and function of CD4^hi Treg by interrupting ICOS-ICOSL interaction with ICOS-Ig. It is found that blockade of ICOS-ICOSL interaction impairs the induction and expansion of CD4^hi Treg induced by allogeneic CD40-activated B cells. More importantly, CD4^hi Treg induced with the addition of ICOS-Ig exhibits decreased suppressive capacity on alloantigen-specific responses. Dysfunction of CD4^hi Treg induced with ICOS-Ig is accompanied with its decreased exocytosis and surface CTLA-4 expression. Through inhibiting endocytosis with E64 and pepstatin A, surface CTLA-4 expression and suppressive functions of induced CD4^hi Treg could be partly reversed. Conclusively, our results demonstrate the beneficial role of ICOS-ICOSL signal pathway in the generation and function of CD4^hi Treg and uncover a novel relationship between ICOS and CTLA-4.     

CD103+CD11b+ Dendritic Cells Induce Th17 T Cells in Muc2-Deficient Mice with Extensively Spread Colitis

Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4⁺ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2^-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103⁺CD11b^- DCs and increased CD103^-CD11b⁺ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1β, IL-6, IL-17, TNFα, and IFNγ combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103⁺CD11b⁺ DCs in the distal colon. CD103⁺CD11b⁺ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells ex vivo. In contrast, CD103^-CD11b⁺ DCs from colitic Muc2^-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103⁺CD11b⁺ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.     

The skewed frequency of B-cell subpopulation CD19+CD24 hiCD38 hi cells in peripheral blood mononuclear cells is correlated with the elevated serum sCD40L in patients with active systemic lupus erythematosus

CD19⁺CD24^hiCD38^hi cells play an essential role in maintaining immune homeostasis. CD40 signaling is involved in regulating the induction and function of CD19⁺CD24^hiCD38^hi cells. Changes in B-cell subpopulations and CD19⁺CD24^hiCD38^hi cells have been observed in systemic lupus erythematosus (SLE) patients. Whether changes in the B-cell subpopulation are related to the aberrant CD40 signaling in SLE patients remains unclear. In this study, we examined changes in the levels of CD19⁺CD24^hiCD38^hi cells and CD19⁺CD24^hiCD38^low cells in peripheral blood mononuclear cells and the serum level of soluble CD40 ligand (sCD40L) in 30 patients with SLE. Through routine biochemical assays and flow cytometry assay, we found that (1) the CD19⁺CD24^hiCD38^hi cell subset was upregulated in SLE patients compared to that in healthy controls (HCs) (P < 0.05); (2) the CD19⁺CD24^hiCD38^low cell subset was downregulated in SLE patients compared with that in HCs; and (3) CD38 expression was positively correlated with SLE manifestations and the serum sCD40L level (P < 0.05). In conclusion, the relative level of Bregs is significantly higher in SLE patients than in HCs and is positively correlated with disease activity and sCD40L level.     

Characterization of Regulatory B Cells in Graves’ Disease and Hashimoto’s Thyroiditis

A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10⁺ B cells. Little is known about the ability of self-antigens to induce IL-10⁺ B cells in Graves’ disease (GD), Hashimoto’s thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10⁺ B-cell differentiation in GD. A similar tendency was observed in healthy donors, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4⁺ T cells. To assess the maximal frequency of inducible IL-10⁺ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10⁺ B-cell frequency was similar in the three groups and correlated with free T₃ levels in GD patients. IL-10⁺ B cells from both patient groups displayed CD25 or TIM-1 more frequently than did those from healthy donors. B-cell expression of two surface marker combinations previously associated with regulatory B-cell functions, CD24^hiCD38^hi and CD27⁺CD43⁺, did not differ between patients and healthy donors. In conclusion, our findings indicate that autoimmune thyroiditis is not associated with reduced frequency of IL-10⁺ B cells. These results do not rule out regulatory B-cell dysfunction, however. The observed phenotypic differences between IL-10⁺ B cells from patients and healthy donors are discussed.     

A Lower Proportion of Regulatory B Cells in Patients with Henoch–Schoenlein Purpura Nephritis

Background

Henoch—Schoenlein purpura is the one of most common types of systemic vasculitis that involves impaired renal function and Henoch-Schoenlein purpura nephritis (HSPN). The diagnosis of this condition is largely based on immunohistologic detection of immunoglobulin A1-containing immune complex in the glomerular deposits of mesangium. Despite clinical advances, the etiopathogenesis of HSPN is still largely unknown.

Methods

In this study, we enrolled 25 newly diagnosed HSPN patients and 14 healthy controls. Then, fractions of B cell subtypes were determined in venous blood using flow cytometry. The serum interleukin (IL)-10 concentration was determined by enzyme-linked immunosorbent assay.

Results

Compared to those in healthy controls, the numbers of CD38⁺CD19⁺, CD86⁺CD19⁺, CD38⁺CD86⁺CD19⁺, and CD95⁺CD19⁺ B cells per microliter of blood were significantly higher in HSPN patients. In contrast, the numbers of CD5⁺CD19⁺, IL-10⁺CD19⁺, CD5⁺CD1d⁺CD19⁺, and IL-10⁺CD5⁺CD1d⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly lower in HSPN patients. Following treatment, the numbers of CD38⁺CD19⁺ and CD86⁺CD19⁺ B cells per microliter of blood were significantly reduced in HSPN patients. However, the numbers of CD5⁺CD1d⁺CD19⁺, CD5⁺CD1d⁺IL-10⁺CD19⁺, and IL-10⁺CD19⁺ B cells per microliter of blood and the serum IL-10 concentration were significantly increased in HSPN patients following treatment. The estimated glomerular filtration rate (eGFR) was negatively correlated with the number of CD38⁺CD19⁺ B cells but positively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration. The 24-h urinary protein concentration was positively correlated with the number of CD38⁺CD19⁺B cells but negatively correlated with the numbers of IL-10⁺CD19⁺, CD1d⁺CD5⁺CD19⁺, and IL-10⁺CD1d⁺CD5⁺CD19⁺B cells per microliter of blood and the serum IL-10 concentration.

Conclusion

Our results suggest that CD38⁺CD19⁺ and CD1d⁺CD5⁺CD19⁺ B cells (Bregs) contribute to the pathogenesis of HSPN.     

Expansion of Parasite-Specific CD4+ and CD8+ T Cells Expressing IL-10 Superfamily Cytokine Members and Their Regulation in Human Lymphatic Filariasis

Background

Lymphatic filariasis (LF) is known to be associated with an increased production of IL-10. The role of the other IL-10 family members in the pathogenesis of infection and/or disease is not known.

Methodology/Principal Findings

We examined the expression patterns of IL-10 family members – IL-19, IL-24 and IL-26 in LF. We demonstrate that both CD4⁺ and CD8⁺ T cells express IL-19, IL-24 and IL-26 and that the frequency of CD4⁺ T cells expressing IL-19 and IL-24 (as well as IL-10) is significantly increased at baseline and following filarial antigen stimulation in patients with LF in comparison to individuals with filarial lymphedema and uninfected individuals. This CD4⁺ T cell expression pattern was associated with increased production of IL-19 and IL-24 by filarial – antigen stimulated PBMC. Moreover, the frequency of CD4⁺ and CD8⁺ T cells expressing IL-26 was significantly increased following filarial antigen stimulation in filarial lymphedema individuals. Interestingly, IL-10 blockade resulted in diminished frequencies of IL-19⁺ and IL-24⁺ T cells, whereas the addition of recombinant IL-10 resulted in significantly increased frequency of IL-19⁺ and IL-24⁺ T cells as well as significantly up regulated IL-19 and IL-24 gene expression, suggesting that IL-10 regulates IL-19 and IL-24 expression in T cells. In addition, IL-1β and IL-23 blockade also induced a diminution in the frequency of IL-19⁺ and IL-24⁺ T cells, indicating a novel role for these cytokines in the induction of IL-19 and IL-24 expressing T cells. Finally, elimination of infection resulted in significantly decreased frequencies of antigen – specific CD4⁺ T cells expressing IL-10, IL-19 and IL-24.

Conclusions

Our findings, therefore, suggest that IL-19 and IL-24 are associated with the regulation of immune responses in active filarial infection and potentially with protection against development of pathology, while IL-26 is predominantly associated with pathology in LF.     

CD4+ T Cell-derived IL-10 Promotes Brucella abortus Persistence via Modulation of Macrophage Function

Evasion of host immune responses is a prerequisite for chronic bacterial diseases; however, the underlying mechanisms are not fully understood. Here, we show that the persistent intracellular pathogen Brucella abortus prevents immune activation of macrophages by inducing CD4⁺CD25⁺ T cells to produce the anti-inflammatory cytokine interleukin-10 (IL-10) early during infection. IL-10 receptor (IL-10R) blockage in macrophages resulted in significantly higher NF-kB activation as well as decreased bacterial intracellular survival associated with an inability of B. abortus to escape the late endosome compartment in vitro. Moreover, either a lack of IL-10 production by T cells or a lack of macrophage responsiveness to this cytokine resulted in an increased ability of mice to control B. abortus infection, while inducing elevated production of pro-inflammatory cytokines, which led to severe pathology in liver and spleen of infected mice. Collectively, our results suggest that early IL-10 production by CD25⁺CD4⁺ T cells modulates macrophage function and contributes to an initial balance between pro-inflammatory and anti-inflammatory cytokines that is beneficial to the pathogen, thereby promoting enhanced bacterial survival and persistent infection.