Two subsets of human marginal zone B cells resolved by global analysis of lymphoid tissues and blood.

B cells generate antibodies that are essential for immune protection, but their subgroups are poorly defined. Here, we perform undirected deep profiling of B cells in matched human lymphoid tissues from deceased transplant organ donors and blood. In addition to identifying unanticipated features of tissue-based B cell differentiation, we resolve two subsets of marginal zone B (MZB) cells differing in cell surface and transcriptomic profiles, clonal relationships to other subsets, enrichment of genes in the NOTCH pathway, distribution bias within splenic marginal zone microenvironment, and immunoglobulin repertoire diversity and hypermutation frequency. Each subset is present in spleen, gut-associated lymphoid tissue, mesenteric lymph nodes, and blood. MZB cells and the lineage from which they are derived are depleted in lupus nephritis. Here, we show that this depletion is of only one MZB subset. The other remains unchanged as a proportion of total B cells compared with health. Thus, it is important to factor MZB cell heterogeneity into studies of human B cell responses and pathology.

Human marginal zone B cell development from early T2 progenitors.

B cells emerge from the bone marrow as transitional (TS) B cells that differentiate through T1, T2, and T3 stages to become naive B cells. We have identified a bifurcation of human B cell maturation from the T1 stage forming IgMhi and IgMlo developmental trajectories. IgMhi T2 cells have higher expression of α4ß7 integrin and lower expression of IL-4 receptor (IL4R) compared with the IgMlo branch and are selectively recruited into gut-associated lymphoid tissue. IgMhi T2 cells also share transcriptomic features with marginal zone B cells (MZBs). Lineage progression from T1 cells to MZBs via an IgMhi trajectory is identified by pseudotime analysis of scRNA-sequencing data. Reduced frequency of IgMhi gut-homing T2 cells is observed in severe SLE and is associated with reduction of MZBs and their putative IgMhi precursors. The collapse of the gut-associated MZB maturational axis in severe SLE affirms its existence in health.

Cryptosporidium parvum Subverts Antimicrobial Activity of CRAMP by Reducing Its Expression in Neonatal Mice.

Cryptosporidium parvum causes diarrhea in infants under 5 years, in immunosuppressed individuals or in young ruminants. This parasite infects the apical side of ileal epithelial cells where it develops itself and induces inflammation. Antimicrobial peptides (AMPs) are part of the innate immune response, playing a major role in the control of the acute phase of C. parvum infection in neonates. Intestinal AMP production in neonates is characterized by high expressions of Cathelicidin Related Antimicrobial Peptide (CRAMP), the unique cathelicidin in mice known to fight bacterial infections. In this study, we investigated the role of CRAMP during cryptosporidiosis in neonates. We demonstrated that sporozoites are sensitive to CRAMP antimicrobial activity. However, during C. parvum infection the intestinal expression of CRAMP was significantly and selectively reduced, while other AMPs were upregulated. Moreover, despite high CRAMP expression in the intestine of neonates at homeostasis, the depletion of CRAMP did not worsen C. parvum infection. This result might be explained by the rapid downregulation of CRAMP induced by infection. However, the exogenous administration of CRAMP dampened the parasite burden in neonates. Taken together these results suggest that C. parvum impairs the production of CRAMP to subvert the host response, and highlight exogenous cathelicidin supplements as a potential treatment strategy.

Resolvin E1 derived from eicosapentaenoic acid prevents hyperinsulinemia and hyperglycemia in a host genetic manner.

Eicosapentaenoic acid (EPA) has garnered attention after the success of the REDUCE-IT trial, which contradicted previous conclusions on EPA for cardiovascular disease risk. Here we first investigated EPA's preventative role on hyperglycemia and hyperinsulinemia. EPA ethyl esters prevented obesity-induced glucose intolerance, hyperinsulinemia, and hyperglycemia in C57BL/6J mice. Supporting NHANES analyses showed that fasting glucose levels of obese adults were inversely related to EPA intake. We next investigated how EPA improved murine hyperinsulinemia and hyperglycemia. EPA overturned the obesity-driven decrement in the concentration of 18-hydroxyeicosapentaenoic acid (18-HEPE) in white adipose tissue and liver. Treatment of obese inbred mice with RvE1, the downstream immunoresolvant metabolite of 18-HEPE, but not 18-HEPE itself, reversed hyperinsulinemia and hyperglycemia through the G-protein coupled receptor ERV1/ChemR23. To translate the findings, we determined if the effects of RvE1 were dependent on host genetics. RvE1's effects on hyperinsulinemia and hyperglycemia were divergent in diversity outbred mice that model human genetic variation. Secondary SNP analyses further confirmed extensive genetic variation in human RvE1/EPA-metabolizing genes. Collectively, the data suggest EPA prevents hyperinsulinemia and hyperglycemia, in part, through RvE1's activation of ERV1/ChemR23 in a host genetic manner. The studies underscore the need for personalized administration of RvE1 based on genetic/metabolic enzyme profiles.

Reduced CD27-IgD- B Cells in Blood and Raised CD27-IgD- B Cells in Gut-Associated Lymphoid Tissue in Inflammatory Bowel Disease.

The intestinal mucosa in inflammatory bowel disease (IBD) contains increased frequencies of lymphocytes and a disproportionate increase in plasma cells secreting immunoglobulin (Ig)G relative to other isotypes compared to healthy controls. Despite consistent evidence of B lineage cells in the mucosa in IBD, little is known of B cell recruitment to the gut in IBD. Here we analyzed B cells in blood of patients with Crohn's disease (CD) and ulcerative colitis (UC) with a range of disease activities. We analyzed the frequencies of known B cell subsets in blood and observed a consistent reduction in the proportion of CD27-IgD- B cells expressing all Ig isotypes in the blood in IBD (independent of severity of disease and treatment) compared to healthy controls. Successful treatment of patients with biologic therapies did not change the profile of B cell subsets in blood. By mass cytometry we demonstrated that CD27-IgD- B cells were proportionately enriched in the gut-associated lymphoid tissue (GALT) in IBD. Since production of TNFα is a feature of IBD relevant to therapies, we sought to determine whether B cells in GALT or the CD27-IgD- subset in particular could contribute to pathology by secretion of TNFα or IL-10. We found that donor matched GALT and blood B cells are capable of producing TNFα as well as IL-10, but we saw no evidence that CD27-IgD- B cells from blood expressed more TNFα compared to other subsets. The reduced proportion of CD27-IgD- B cells in blood and the increased proportion in the gut implies that CD27-IgD- B cells are recruited from the blood to the gut in IBD. CD27-IgD- B cells have been implicated in immune responses to intestinal bacteria and recruitment to GALT, and may contribute to the intestinal inflammatory milieu in IBD.

Frontline Science: A reduction in DHA-derived mediators in male obesity contributes toward defects in select B cell subsets and circulating antibody.

Obesity dysregulates B cell populations, which contributes toward poor immunological outcomes. We previously reported that differing B cell subsets are lowered in the bone marrow of obese male mice. Here, we focused on how lipid metabolites synthesized from docosahexaenoic acid (DHA) known as specialized pro-resolving lipid mediators (SPMs) influence specific B cell populations in obese male mice. Metabololipidomics revealed that splenic SPM precursors 14-hydroxydocosahexaenoic acid (14-HDHA), 17-hydroxydocosahexaenoic acid (17-HDHA), and downstream protectin DX (PDX) were decreased in obese male C57BL/6J mice. Simultaneous administration of these mediators to obese mice rescued major decrements in bone marrow B cells, modest impairments in the spleen, and circulating IgG2c, which is pro-inflammatory in obesity. In vitro studies with B cells, flow cytometry experiments with ALOX5-/- mice, and lipidomic analyses revealed the lowering of 14-HDHA/17-HDHA/PDX and dysregulation of B cell populations in obesity was driven indirectly via B cell extrinsic mechanisms. Notably, the lowering of lipid mediators was associated with an increase in the abundance of n-6 polyunsaturated fatty acids, which have a high affinity for SPM-generating enzymes. Subsequent experiments revealed female obese mice generally maintained the levels of SPM precursors, B cell subsets, and antibody levels. Finally, obese human females had increased circulating plasma cells accompanied by ex vivo B cell TNFα and IL-10 secretion. Collectively, the data demonstrate that DHA-derived mediators of the SPM pathway control the number of B cell subsets and pro-inflammatory antibody levels in obese male but not female mice through a defect that is extrinsic to B cells.

Spatiotemporal segregation of human marginal zone and memory B cell populations in lymphoid tissue.

Human memory B cells and marginal zone (MZ) B cells share common features such as the expression of CD27 and somatic mutations in their IGHV and BCL6 genes, but the relationship between them is controversial. Here, we show phenotypic progression within lymphoid tissues as MZ B cells emerge from the mature naïve B cell pool via a precursor CD27-CD45RBMEM55+ population distant from memory cells. By imaging mass cytometry, we find that MZ B cells and memory B cells occupy different microanatomical niches in organised gut lymphoid tissues. Both populations disseminate widely between distant lymphoid tissues and blood, and both diversify their IGHV repertoire in gut germinal centres (GC), but nevertheless remain largely clonally separate. MZ B cells are therefore not developmentally contiguous with or analogous to classical memory B cells despite their shared ability to transit through GC, where somatic mutations are acquired.

Effects of fish oils on ex vivo B-cell responses of obese subjects upon BCR/TLR stimulation: a pilot study.

The long-chain n-3 polyunsaturated fatty acids (LC-PUFAs) eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) in fish oil have immunomodulatory properties. B cells are a poorly studied target of EPA/DHA in humans. Therefore, in this pilot study, we tested how n-3 LC-PUFAs influence B-cell responses of obese humans. Obese men and women were assigned to consume four 1-g capsules per day of olive oil (OO, n=12), fish oil (FO, n=12) concentrate or high-DHA-FO concentrate (n=10) for 12 weeks in a parallel design. Relative to baseline, FO (n=9) lowered the percentage of circulating memory and plasma B cells, whereas the other supplements had no effect. There were no postintervention differences between the three supplements. Next, ex vivo B-cell cytokines were assayed after stimulation of Toll-like receptors (TLRs) and/or the B-cell receptor (BCR) to determine if the effects of n-3 LC-PUFAs were pathway-dependent. B-cell IL-10 and TNFα secretion was respectively increased with high DHA-FO (n=10), relative to baseline, with respective TLR9 and TLR9+BCR stimulation. OO (n=12) and FO (n=12) had no influence on B-cell cytokines compared to baseline, and there were no differences in postintervention cytokine levels between treatment groups. Finally, ex vivo antibody levels were assayed with FO (n=7) after TLR9+BCR stimulation. Compared to baseline, FO lowered IgM but not IgG levels accompanied by select modifications to the plasma lipidome. Altogether, the results suggest that n-3 LC-PUFAs could modulate B-cell activity in humans, which will require further testing in a larger cohort.

B Cell Activity Is Impaired in Human and Mouse Obesity and Is Responsive to an Essential Fatty Acid upon Murine Influenza Infection.

Obesity is associated with increased risk for infections and poor responses to vaccinations, which may be due to compromised B cell function. However, there is limited information about the influence of obesity on B cell function and underlying factors that modulate B cell responses. Therefore, we studied B cell cytokine secretion and/or Ab production across obesity models. In obese humans, B cell IL-6 secretion was lowered and IgM levels were elevated upon ex vivo anti-BCR/TLR9 stimulation. In murine obesity induced by a high fat diet, ex vivo IgM and IgG were elevated with unstimulated B cells. Furthermore, the high fat diet lowered bone marrow B cell frequency accompanied by diminished transcripts of early lymphoid commitment markers. Murine B cell responses were subsequently investigated upon influenza A/Puerto Rico/8/34 infection using a Western diet model in the absence or presence of docosahexaenoic acid (DHA). DHA, an essential fatty acid with immunomodulatory properties, was tested because its plasma levels are lowered in obesity. Relative to controls, mice consuming the Western diet had diminished Ab titers whereas the Western diet plus DHA improved titers. Mechanistically, DHA did not directly target B cells to elevate Ab levels. Instead, DHA increased the concentration of the downstream specialized proresolving lipid mediators (SPMs) 14-hydroxydocosahexaenoic acid, 17-hydroxydocosahexaenoic acid, and protectin DX. All three SPMs were found to be effective in elevating murine Ab levels upon influenza infection. Collectively, the results demonstrate that B cell responses are impaired across human and mouse obesity models and show that essential fatty acid status is a factor influencing humoral immunity, potentially through an SPM-mediated mechanism.

Oncostatin M overexpression induces skin inflammation but is not required in the mouse model of imiquimod-induced psoriasis-like inflammation.

Oncostatin M (OSM) has been reported to be overexpressed in psoriasis skin lesions and to exert proinflammatory effects in vitro on human keratinocytes. Here, we report the proinflammatory role of OSM in vivo in a mouse model of skin inflammation induced by intradermal injection of murine OSM-encoding adenovirus (AdOSM) and compare with that induced by IL-6 injection. Here, we show that OSM potently regulates the expression of genes involved in skin inflammation and epidermal differentiation in murine primary keratinocytes. In vivo, intradermal injection of AdOSM in mouse ears provoked robust skin inflammation with epidermal thickening and keratinocyte proliferation, while minimal effect was observed after AdIL-6 injection. OSM overexpression in the skin increased the expression of the S100A8/9 antimicrobial peptides, CXCL3, CCL2, CCL5, CCL20, and Th1/Th2 cytokines, in correlation with neutrophil and macrophage infiltration. In contrast, OSM downregulated the expression of epidermal differentiation genes, such as cytokeratin-10 or filaggrin. Collectively, these results support the proinflammatory role of OSM when it is overexpressed in the skin. However, OSM expression was not required in the murine model of psoriasis induced by topical application of imiquimod, as demonstrated by the inflammatory phenotype of OSM-deficient mice or wild-type mice treated with anti-OSM antibodies.

CCL20 Displays Antimicrobial Activity Against Cryptosporidium parvum, but Its Expression Is Reduced During Infection in the Intestine of Neonatal Mice.

CCL20 is a chemokine with antimicrobial activity. We investigated its expression and role during neonatal cryptosporidiosis, a worldwide protozoan enteric disease leading to severe diarrhea. Surprisingly, during infection by Cryptosporidium parvum, CCL20 production by the intestine of neonatal mice is reduced by a mechanism independent both of the enteric flora and of interferon γ, a key cytokine for the resolution of this infection. However, oral administration of recombinant CCL20 to neonatal mice significantly reduced the parasite load by a mechanism that was independent of immune cell recruitment and occurred instead by direct cytolytic activity on free stages of the parasite. MiR21 functionally targets CCL20 and is upregulated during the infection, thus contributing to the downregulation of the chemokine. Our findings demonstrate for the first time the direct antiparasitic activity of CCL20 against an enteric protozoan and its downregulation during C. parvum infection, which is detrimental to parasite clearance.

The gut flora is required for the control of intestinal infection by poly(I:C) administration in neonates.

We found that immunostimulation of the intestinal immune system of neonatal mice by poly(I:C) injection decreased intestinal infection by the parasite Cryptosporidium parvum. We showed that the presence of dendritic cells and the cooperation of mutually dependent cytokines, such as IL-12p40, and type I and type II IFNs, were involved in the mechanism of protection induced by poly(I:C). This protection is dependent not only on TLR3-TRIF signaling, but also on the activation of the TLR5-MyD88 pathway by gut microbiota. These results raise the possibility that flagellated intestinal commensal bacteria may, in the presence of natural or synthetic agonists of TLR3, provide synergy between the TRIF and MyD88 signaling pathways, thereby favoring the development of mucosal defenses. In this addendum, we summarize these recent findings and discuss their implications for neonatal infections and immunomodulatory strategies.

Poly(I:C)-induced protection of neonatal mice against intestinal Cryptosporidium parvum infection requires an additional TLR5 signal provided by the gut flora.

The neonatal intestinal immune system is still undergoing development at birth, leading to a higher susceptibility to mucosal infections. In this study, we investigated the effect of poly(I:C) on controlling enteric infection by the protozoan Cryptosporidium parvum in neonatal mice. After poly(I:C) administration, a rapid reduction in parasite burden was observed and proved to be dependent on CD11c(+) cells and TLR3/TRIF signaling. Protection against C. parvum required additional signals provided by the gut flora through TLR5 and MyD88 signaling. This cooperation gave rise to higher levels of expression of critical mutually dependent cytokines such as interleukin 12p40 and type 1 and type 2 interferons, the last 2 being known to play a key role in the elimination of infected enterocytes. Our findings demonstrate in neonatal mice how gut flora synergizes with poly(I:C) to elicit protective intestinal immunity against an intracellular pathogen.

Intestinal CD103+ dendritic cells are key players in the innate immune control of Cryptosporidium parvum infection in neonatal mice.

Cryptosporidium parvum is a zoonotic protozoan parasite found worldwide, that develops only in the gastrointestinal epithelium and causes profuse diarrhea. Using a mouse model of C. parvum infection, we demonstrated by conditional depletion of CD11c+ cells that these cells are essential for the control of the infection both in neonates and adults. Neonates are highly susceptible to C. parvum but the infection is self-limited, whereas adults are resistant unless immunocompromised. We investigated the contribution of DC to the age-dependent susceptibility to infection. We found that neonates presented a marked deficit in intestinal CD103+ DC during the first weeks of life, before weaning, due to weak production of chemokines by neonatal intestinal epithelial cells (IEC). Increasing the number of intestinal CD103+ DC in neonates by administering FLT3-L significantly reduced susceptibility to the infection. During infections in neonates, the clearance of the parasite was preceded by a rapid recruitment of CD103+ DC mediated by CXCR3-binding chemokines produced by IEC in response to IFNγ. In addition to this key role in CD103+ DC recruitment, IFNγ is known to inhibit intracellular parasite development. We demonstrated that during neonatal infection CD103+ DC produce IL-12 and IFNγ in the lamina propria and the draining lymph nodes. Thus, CD103+DC are key players in the innate immune control of C. parvum infection in the intestinal epithelium. The relative paucity of CD103+ DC in the neonatal intestine contributes to the high susceptibility to intestinal infection.