P2RX7 gene variants associate with altered inflammasome assembly and reduced pyroptosis in chronic nonbacterial osteomyelitis (CNO).

Chronic nonbacterial osteomyelitis (CNO), an autoinflammatory bone disease primarily affecting children, can cause pain, hyperostosis and fractures, affecting quality-of-life and psychomotor development. This study investigated CNO-associated variants in P2RX7, encoding for the ATP-dependent trans-membrane K+ channel P2X7, and their effects on NLRP3 inflammasome assembly. Whole exome sequencing in two related transgenerational CNO patients, and target sequencing of P2RX7 in a large CNO cohort (N = 190) were conducted. Results were compared with publicly available datasets and regional controls (N = 1873). Findings were integrated with demographic and clinical data. Patient-derived monocytes and genetically modified THP-1 cells were used to investigate potassium flux, inflammasome assembly, pyroptosis, and cytokine release. Rare presumably damaging P2RX7 variants were identified in two related CNO patients. Targeted P2RX7 sequencing identified 62 CNO patients with rare variants (32.4%), 11 of which (5.8%) carried presumably damaging variants (MAF <1%, SIFT "deleterious", Polyphen "probably damaging", CADD >20). This compared to 83 of 1873 controls (4.4%), 36 with rare and presumably damaging variants (1.9%). Across the CNO cohort, rare variants unique to one (Median: 42 versus 3.7) or more (≤11 patients) participants were over-represented when compared to 190 randomly selected controls. Patients with rare damaging variants more frequently experienced gastrointestinal symptoms and lymphadenopathy while having less spinal, joint and skin involvement (psoriasis). Monocyte-derived macrophages from patients, and genetically modified THP-1-derived macrophages reconstituted with CNO-associated P2RX7 variants exhibited altered potassium flux, inflammasome assembly, IL-1ß and IL-18 release, and pyroptosis. Damaging P2RX7 variants occur in a small subset of CNO patients, and rare P2RX7 variants may represent a CNO risk factor. Observations argue for inflammasome inhibition and/or cytokine blockade and may allow future patient stratification and individualized care.

[PAPA syndrome with Crohn's disease and primary sclerosing cholangitis/autoimmune hepatitis overlap syndrome]. / PAPA-Syndrom mit Morbus Crohn und primär sklerosierender Cholangitis/Autoimmunhepatitis-Overlap-Syndrom.

BACKGROUND: The PAPA syndrome, an acronym for pyogenic sterile arthritis, pyoderma gangraenosum and acne, is an autosomal dominant hereditary disease which is caused by a mutation in the PSTPIP1 ("proline-serine-threonine phosphatase interacting protein 1") gene located on chromosome 15 and encodes the proline-serine-threonine phosphatase-interacting protein 1. An association with Crohn's disease (CD), autoimmune diseases of the liver and PAPA syndrome has not yet been reported in the literature. OBJECTIVE: To thoroughly investigate a family with three affected members (mother and 2 children) with newly diagnosed PAPA syndrome and intestinal and hepatobiliary symptoms. MATERIAL AND METHODS: We performed an in-depth phenotyping, dermatologic, radiologic, rheumatologic, gastroenterologic, histologic and genetic analysis in this family. RESULTS: All three family members could be newly diagnosed as suffering from PAPA syndrome and carried the known disease-causing mutation c.688G > A (p.Ala230Thr) in the PSTPIP1 gene. The younger son suffered from CD in addition to PAPA syndrome. The mother additionally suffered from ulcerative colitis (UC) and an overlap syndrome between autoimmune hepatitis (AIH) and primary sclerosing cholangitis (PSC). A mutation in in the NOD2 ("nucleotide binding oligomerization domain containing protein 2") gene could not be detected in any of the three persons affected. CONCLUSION: We extended the symptoms of PAPA syndrome to CD and autoimmune liver disease. These different disease entities might share a similar pathogenetic mechanism or even represent a new syndrome. This can be clarified in the future by screening patients with PAPA syndrome for intestinal and also hepatobiliary diseases.

Signatures of T and B Cell Development, Functional Responses and PD-1 Upregulation After HCMV Latent Infections and Reactivations in Nod.Rag.Gamma Mice Humanized With Cord Blood CD34+ Cells.

Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the Gaussia luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. Foci of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14+, CD169+, and CD34+ cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4+ T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG+ plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4+ T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel in vivo humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.

Human Basophils Modulate Plasma Cell Differentiation and Maturation.

Basophils represent <1% of circulating leukocytes. They play a crucial role during allergy and helminth-induced Th2 responses. However, recent data also suggest a contribution to the pathogenesis of autoimmune diseases. Basophils from patients with systemic lupus erythematosus show an activated phenotype, correlating to disease activity. Furthermore, murine basophils or their mediators enhance memory responses and plasma cell (PC) survival, suggesting that they directly modulate the function of B cells. This is highly relevant with respect to human allergy and autoimmunity because a possible modulation of B cell differentiation by basophils could point to new therapeutic targets. Therefore, the interaction between human B cells and basophils and the mechanism underlying this interaction were investigated in detail. Using two different methods to induce PC differentiation, we found that human basophils enhance B cell proliferation, class switching, differentiation into PC, maturation of PC, and production of Igs, especially IgG. Basophil supernatants enhanced the expression of the B cell markers CD23 and CD40, which are important for B cell differentiation into IgG-producing PC. This was mainly IL-4 dependent. IL-3 amplified the number of PC in vitro, and acted synergistically with basophils in enhancing Ab production. Thus, human basophils modulate B cell differentiation into Ab-producing PC. Their contribution as modulators and effectors during allergy and autoimmunity should be considered when designing new therapeutic options.

B cell subsets are modulated during allergic airway inflammation but are not required for the development of respiratory tolerance in a murine model.

Allergic asthma is a widespread chronic inflammatory disease of the airways. The role of different B cell subsets in developing asthma and respiratory tolerance is not well known. Especially regulatory B (Breg) cells are proposed to be important in asthma regulation. Using wild-type (WT) and B cell-deficient (µMT) mice we investigated how B cells are affected by induction of allergic airway inflammation and respiratory tolerance and whether they are necessary to develop these conditions. WT mice with an asthma-like phenotype, characterized by increased airway hyper reactivity, eosinophilic airway inflammation, mucus hypersecretion and elevated Th2 cytokines, exhibited increased MHCII and CD23 expression on follicular mature B cells in lung, bronchial lymph nodes (bLN) and spleen, which contributed to allergen-specific T cell proliferation in vitro. Germinal center B cell numbers were elevated and associated with increased production of allergen-specific immunoglobulins especially in bLN. In contrast, respiratory tolerance clearly attenuated these B cell alterations and directly enhanced marginal zone precursor B cells, which induced regulatory T cells in vitro. However, µMT mice developed asthma-like and tolerized phenotypes like WT mice. Our data indicate that although B cell subsets are affected by asthma-like and respiratory tolerant phenotypes, B cells are not required for tolerance induction.

Reduced Number of Transitional and Naive B Cells in Addition to Decreased BAFF Levels in Response to the T Cell Independent Immunogen Pneumovax®23.

Protective immunity against T cell independent (TI) antigens such as Streptococcus pneumoniae is characterized by antibody production of B cells induced by the combined activation of T cell independent type 1 and type 2 antigens in the absence of direct T cell help. In mice, the main players in TI immune responses have been well defined as marginal zone (MZ) B cells and B-1 cells. However, the existence of human equivalents to these B cell subsets and the nature of the human B cell compartment involved in the immune reaction remain elusive. We therefore analyzed the effect of a TI antigen on the B cell compartment through immunization of healthy individuals with the pneumococcal polysaccharide (PnPS)-based vaccine Pneumovax®23, and subsequent characterization of B cell subpopulations. Our data demonstrates a transient decrease of transitional and naïve B cells, with a concomitant increase of IgA+ but not IgM+ or IgG+ memory B cells and a predominant generation of PnPS-specific IgA+ producing plasma cells. No alterations could be detected in T cells, or proposed human B-1 and MZ B cell equivalents. Consistent with the idea of a TI immune response, antigen-specific memory responses could not be observed. Finally, BAFF, which is supposed to drive class switching to IgA, was unexpectedly found to be decreased in serum in response to Pneumovax®23. Our results demonstrate that a characteristic TI response induced by Pneumovax®23 is associated with distinct phenotypical and functional changes within the B cell compartment. Those modulations occur in the absence of any modulations of T cells and without the development of a specific memory response.

B-1 cells as a source of IgA.

Immunoglobulin A (IgA) is the most abundantly produced immunoglobulin found primarily on mucosal surfaces. The generation of IgA and its involvement in mucosal immune responses have been intensely studied over the past years. IgA can be generated in T cell-dependent and T cell-independent pathways, and it has an important impact on maintaining homeostasis within the mucosal immune system. There is good evidence that B-1 cells contribute substantially to the production of mucosal IgA and thus play an important role in regulating commensal microbiota. However, whether B-1 cells produce antigen-specific or only nonspecific IgA remains to be determined. This review will discuss what is currently known about IgA production by B-1 cells and the functional relevance of B-1 cell-derived IgA both in vitro and in vivo.

Distinct effects of methotrexate and etanercept on the B cell compartment in patients with juvenile idiopathic arthritis.

OBJECTIVE: B cells have been shown to play an important role in the pathogenesis of rheumatoid arthritis and juvenile idiopathic arthritis (JIA). Current treatments include the disease-modifying antirheumatic drugs methotrexate (MTX) and tumor necrosis factor α inhibition with etanercept. This study was undertaken to determine how these drugs influence the B cell compartment in patients with JIA. METHODS: B cell subpopulations and follicular helper T (Tfh) cells in the peripheral blood of JIA patients were investigated by multicolor flow cytometry. Serum immunoglobulin and BAFF levels were determined by enzyme-linked immunosorbent assay. RESULTS: There was a significant decrease in transitional B cells and significantly lower serum immunoglobulin levels in patients receiving MTX than in untreated patients and those receiving etanercept. In contrast, etanercept treatment had no effect on most of the B cell subpopulations, but resulted in significantly lower BAFF levels and increased numbers of Tfh cells. Thus, our findings indicate an unexpected and previously unknown direct effect of low-dose MTX on B cells, whereas etanercept had a more indirect influence. CONCLUSION: Our results contribute to a better understanding of the potency of MTX in autoantibody-mediated autoimmune disease and present a possible mechanism of prevention of the development of drug-induced antibodies to biologic agents. The finding that MTX and etanercept affect the B cell compartment differently supports the notion that combination therapy with etanercept and MTX is more effective than monotherapy.

Altered B cell homeostasis is associated with type I diabetes and carriers of the PTPN22 allelic variant.

The PTPN22 genetic variant 1858T, encoding Lyp620W, is associated with multiple autoimmune disorders for which the production of autoantibodies is a common feature, suggesting a loss of B cell tolerance. Lyp620W results in blunted BCR signaling in memory B cells. Because BCR signal strength is tightly coupled to central and peripheral tolerance, we examined whether Lyp620W impacts peripheral B cell homeostasis in healthy individuals heterozygous for the PTPN221858T variant. We found that these subjects display alterations in the composition of the B cell pool that include specific expansion of the transitional and anergic IgD(+)IgM(-)CD27(-) B cell subsets. The PTPN22 1858T variant was further associated with significantly diminished BCR signaling and a resistance to apoptosis in both transitional and naive B cells. Strikingly, parallel changes in both BCR signaling and composition of B cell compartment were observed in type 1 diabetic subjects, irrespective of PTPN22 genotype, revealing a novel immune phenotype and likely shared mechanisms leading to a loss of B cell tolerance. Our combined findings suggest that Lyp620W-mediated effects, due in part to the altered BCR signaling threshold, contribute to breakdown of peripheral tolerance and the entry of autoreactive B cells into the naive B cell compartment.

WASp-deficient B cells play a critical, cell-intrinsic role in triggering autoimmunity.

Patients with the immunodeficiency Wiskott-Aldrich syndrome (WAS) frequently develop systemic autoimmunity. Here, we demonstrate that mutation of the WAS gene results in B cells that are hyperresponsive to B cell receptor and Toll-like receptor (TLR) signals in vitro, thereby promoting a B cell-intrinsic break in tolerance. Whereas this defect leads to autoantibody production in WAS protein-deficient (WASp(-/-)) mice without overt disease, chimeric mice in which only the B cell lineage lacks WASp exhibit severe autoimmunity characterized by spontaneous germinal center formation, class-switched autoantibodies, renal histopathology, and early mortality. Both T cell help and B cell-intrinsic TLR engagement play important roles in promoting disease in this model, as depletion with anti-CD4 antibodies or generation of chimeric mice with B cells deficient in both WASp and MyD88 prevented development of autoimmune disease. These data highlight the potentially harmful role for cell-intrinsic loss of B cell tolerance in the setting of normal T cell function, and may explain why WAS patients with mixed chimerism after stem cell transplantation often develop severe humoral autoimmunity.

Reduced c-myc expression levels limit follicular mature B cell cycling in response to TLR signals.

The splenic B cell compartment is comprised of two major, functionally distinct, mature B cell subsets, i.e., follicular mature (FM) and marginal zone (MZ) B cells. Whereas MZ B cells exhibit a robust proliferative response following stimulation with the TLR4 ligand LPS, FM B cells display markedly delayed and reduced levels of proliferation to the identical stimulus. The current study was designed to identify a potential mechanism(s) accounting for this differential responsiveness. In contrast to the delay in cell cycle entry, FM and MZ B cells exhibited nearly identical LPS-driven alterations in the expression level of cell surface activation markers. Furthermore, both the NF-kappaB and mTOR signaling cascades were similarly activated by LPS stimulation in FM vs MZ B cells, while inducible activation of ERK and AKT were nearly absent in both subsets. MZ B cells, however, exhibited higher basal levels of phospho-AKT and pS6, consistent with a preactivated status. Importantly, both basal and LPS activation-induced c-myc expression was markedly reduced in FM vs MZ B cells and enforced c-myc expression fully restored the defective proliferative response in FM B cells. These data support a model wherein TLR responses in FM B cells are tightly regulated by limiting c-myc levels, thereby providing an important checkpoint to control nonspecific FM B cell activation in the absence of cognate Ag.

Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis.

To more precisely identify the B-cell phenotype in Wiskott-Aldrich syndrome (WAS), we used 3 distinct murine in vivo models to define the cell intrinsic requirements for WAS protein (WASp) in central versus peripheral B-cell development. Whereas WASp is dispensable for early bone marrow B-cell development, WASp deficiency results in a marked reduction in each of the major mature peripheral B-cell subsets, exerting the greatest impact on marginal zone and B1a B cells. Using in vivo bromodeoxyuridine labeling and in vitro functional assays, we show that these deficits reflect altered peripheral homeostasis, partially resulting from an impairment in integrin function, rather than a developmental defect. Consistent with these observations, we also show that: (1) WASp expression levels increase with cell maturity, peaking in those subsets exhibiting the greatest sensitivity to WASp deficiency; (2) WASp(+) murine B cells exhibit a marked selective advantage beginning at the late transitional B-cell stage; and (3) a similar in vivo selective advantage is manifest by mature WASp(+) human B cells. Together, our data provide a better understanding of the clinical phenotype of WAS and suggest that gene therapy might be a useful approach to rescue altered B-cell homeostasis in this disease.

Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation.

We have characterized a distinct, late transitional B cell subset, CD21(int) transitional 2 (T2) B cells. In contrast to early transitional B cells, CD21(int) T2 B cells exhibit augmented responses to a range of potential microenvironmental stimuli. Adoptive transfer studies demonstrate that this subset is an immediate precursor of both follicular mature and marginal zone (MZ) B cells. In vivo, a large percentage of CD21(int) T2 B cells has entered the cell cycle, and the cycling subpopulation exhibits further augmentation in mitogenic responses and B cell-activating factor of the TNF family (BAFF) receptor expression. Consistent with these features, CD21(int) T2 cells exhibit preferential responses to BAFF-facilitated homeostatic signals in vivo. In addition, we demonstrate that M167 B cell receptor (BCR) idiotypic-specific B cells are first selected within the cycling CD21(int) T2 population, ultimately leading to preferential enrichment of these cells within the MZ B cell compartment. These data, in association with the coordinate role for BAFF and microenvironmental cues in determining the mature BCR repertoire, imply that this subset functions as a unique selection point in peripheral B cell development.

Novel suppressive function of transitional 2 B cells in experimental arthritis.

The immune system contains natural regulatory cells important in the maintenance of tolerance. Although this suppressive function is usually attributed to CD4 regulatory T cells, recent reports have revealed an immunoregulatory role for IL-10-producing B cells in the context of several autoimmune diseases including collagen-induced arthritis. In the present study, we attribute this suppressive function to a B cell subset expressing high levels of CD21, CD23, and IgM, previously identified as transitional 2-marginal zone precursor (T2-MZP) B cells. T2-MZP B cells are present in the spleens of naive mice and increase during the remission phase of arthritis. Following adoptive transfer to immunized DBA/1 mice, T2-MZP B cells significantly prevented new disease and ameliorated established disease. The suppressive effect on arthritis was paralleled by an inhibition of Ag-specific T cell activation and a reduction in cells exhibiting Th1-type functional responses. We also provide evidence that this regulatory subset mediates its suppression through the secretion of suppressive cytokines and not by cell-to-cell contact. The ability to regulate an established immune response by T2-MZP B cells endows this subset of B cells with a striking and previously unrecognized immunoregulatory potential.