The avidity of PR3‐ANCA in patients with granulomatosis with polyangiitis during follow‐up

The objective of this study is to investigate whether the avidity of proteinase‐3‐anti‐neutrophil cytoplasmic antibody (PR3‐ANCA) changes during follow‐up in different subgroups of patients with granulomatosis with polyangiitis (GPA). We selected 10 patients with renal relapsing GPA, 10 patients with renal non‐relapsing GPA and 10 patients with non‐renal relapsing GPA. In all patients, an ANCA rise occurred during remission. The avidity was measured using a chaotropic approach at the time of an ANCA rise and at the time of a relapse in relapsing patients or time‐matched during remission in non‐relapsing patients. No difference was observed in the avidity at the ANCA rise between renal relapsing patients [26·2% (15·5–47·5)], renal patients without a relapse [39·6% (21·2–63·4)] and non‐renal relapsing patients [34·2% (21·6–59·5)]. In renal relapsing patients, the avidity increased significantly from the moment of the ANCA rise to the relapse [difference 6·4% (0·0–17·1), P = 0·0273]. The avidity did not increase after an ANCA rise in renal non‐relapsing patients [difference 3·5 (?6·0 to 10·1), P = 0·6250] or in non‐renal relapsing patients [difference ?3·1% (?8·0 to 5·0), P = 0·5703]. The avidity of PR3‐ANCA increases after an ANCA rise during follow‐up in renal relapsing patients, but not after an ANCA rise in renal patients who remain in remission or in non‐renal relapsing patients.     

Neutrophils from vasculitis patients exhibit an increased propensity for activation by anti‐neutrophil cytoplasmic antibodies

Anti‐neutrophil cytoplasmic antibodies (ANCA) are thought to be pathogenic in ANCA‐associated vasculitis (AAV) by stimulating polymorphonuclear leucocytes (PMNs) to degranulate and produce reactive oxygen species (ROS). The aim of this study was to investigate if PMNs from AAV patients are stimulated more readily by ANCA compared with PMNs from healthy controls (HCs). Differences in ANCA characteristics that can account for different stimulation potential were also studied. PMNs from five AAV patients and five HCs were stimulated with 10 different immunoglobulins (Ig)Gs, purified from PR3–ANCA‐positive patients, and ROS production, degranulation and neutrophil extracellular trap (NET) formation was measured. ANCA levels, affinity and clinical data of the AAV donors were recorded. The results show that PMNs from AAV patients produce more intracellular ROS (P = 0·019), but degranulate to a similar extent as PMNs from HCs. ROS production correlated with NET formation. Factors that may influence the ability of ANCA to activate PMNs include affinity and specificity for N‐terminal epitopes. In conclusion, our results indicate that PMNs from AAV patients in remission behave quite similarly to HC PMNs, with the exception of a greater intracellular ROS production. This could contribute to more extensive NET formation and thus an increased exposure of the ANCA autoantigens to the immune system.     

Alterations in circulating lymphoid cell populations in systemic small vessel vasculitis are non‐specific manifestations of renal injury

Innate lymphocyte populations, such as innate lymphoid cells (ILCs), γδ T cells, invariant natural killer T (iNK T) cells and mucosal‐associated invariant T (MAIT) cells are emerging as important effectors of innate immunity and are involved in various inflammatory and autoimmune diseases. The aim of this study was to assess the frequencies and absolute numbers of innate lymphocytes as well as conventional lymphocytes and monocytes in peripheral blood from a cohort of anti‐neutrophil cytoplasm autoantibody (ANCA)‐associated vasculitis (AAV) patients. Thirty‐eight AAV patients and 24 healthy and disease controls were included in the study. Patients with AAV were sampled both with and without immunosuppressive treatment, and in the setting of both active disease and remission. The frequencies of MAIT and ILC2 cells were significantly lower in patients with AAV and in the disease control group compared to healthy controls. These reductions in the AAV patients remained during remission. B cell count and frequencies were significantly lower in AAV in remission compared to patients with active disease and disease controls. Despite the strong T helper type 2 (Th) preponderance of eosinophilic granulomatosis with polyangiitis, we did not observe increased ILC2 frequency in this cohort of patients. The frequencies of other cell types were similar in all groups studied. Reductions in circulating ILC2 and MAIT cells reported previously in patients with AAV are not specific for AAV, but are more likely to be due to non‐specific manifestations of renal impairment and chronic illness. Reduction in B cell numbers in AAV patients experiencing remission is probably therapy‐related.     

The expression of Toll‐like receptors 2, 4 and 9 in kidneys of patients with anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis

Increasing evidence suggested that Toll-like receptors (TLRs) were critically involved in immune responses of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). The current study aimed to investigate the expression of TLR-2, TLR-4 and TLR-9 in kidneys of patients with ANCA-associated vasculitis. Renal biopsy specimens were collected from 24 patients with AAV. The expression of TLR-2, TLR-4 and TLR-9 in kidneys was detected by immunohistochemistry. Double immunofluorescence staining was performed to detect the expression of TLRs on various kinds of cells. In renal specimens, immunohistochemical examination revealed that expression of TLR-2 and TLR-4 could be detected in the glomeruli of AAV patients, while TLR-2 and TLR-4 were scarcely detected in the glomeruli of normal controls. Double immunofluorescence staining of TLR-2, TLR-4 and CD31 indicated that TLR-4 and TLR-2 were expressed on endothelial cells in the glomeruli. In the tubulointerstitial compartment, expression of TLR-2, TLR-4 and TLR-9 could be detected in both AAV patients and normal controls. The mean optical density of TLR-2 and TLR-4 in the tubulointerstitial compartment in AAV patients were significantly higher than that in normal controls. Among AAV patients, correlation analysis showed that the mean optical density of TLR-4 in the glomeruli correlated inversely with the initial serum creatinine, the proportion of total crescents and the proportion of cellular crescents in renal specimens (r = −0·419, P = 0·041; r = −0·506, P = 0·012; r = −0·505, P = 0·012, respectively). The expression of TLR-2 and TLR-4 was dysregulated in kidneys of AAV patients. The expression of TLR-4 in glomeruli was associated with the severity of renal injury.     

Skewing toward Treg and Th2 responses is a characteristic feature of sustained remission in ANCA‐positive granulomatosis with polyangiitis

The objective of our study was to evaluate the T‐helper (Th) and regulatory T (Treg) cell profile in ANCA‐positive granulomatosis with polyangiitis (GPA) and its relation to disease activity. In a prospective study, we studied two groups of GPA patients: (i) disease flare (active‐GPA, BVAS>6, n = 19), (ii) sustained remission (≥ 1‐year prior enrollment, inactive‐GPA, BVAS = 0, n = 18). 24 age‐sex matched healthy subjects served as controls. Active‐GPA patients were followed for 6 months and reevaluated during remission (early remission; n = 13). We analyzed subsets of Th‐cells (flow cytometry), production of signature cytokines by in vitro stimulated lymphocytes, and broad spectrum of serum cytokines (Luminex). In all GPA patients we observed expansion of effector Th17 cells, and increased production of IL‐17A by in vitro stimulated T cells, as compared to controls. Disease flare was characterized by marked reduction in Treg cells, whereas in sustained remission we showed expansion of both Treg and Th2 subset. Finally, analyzing the cytokine profile, we identified CCL23 and LIGHT, as potential biomarkers of active disease. We conclude that in GPA, expansion of Treg and Th2 lymphocytes in parallel to increased Th17 response is a characteristic feature of sustained remission. In contrast, Treg cells are markedly decreased in disease flare.     

CD14 expression is increased on monocytes in patients with anti‐neutrophil cytoplasm antibody (ANCA)‐associated vasculitis and correlates with the expression of ANCA autoantigens

Monocyte subsets with differing functional properties have been defined by their expression of CD14 and CD16. We investigated these subsets in anti‐neutrophil cytoplasm antibody (ANCA)‐associated vasculitis (AAV) and determined their surface expression of ANCA autoantigens. Flow cytometry was performed on blood from 14 patients with active AAV, 46 patients with AAV in remission and 21 controls. The proportion of classical (CD14^highCD16^neg/low), intermediate (CD14^highCD16^high) and non‐classical (CD14^lowCD16^high) monocytes and surface expression levels of CD14 and CD16 were determined, as well as surface expression of proteinase 3 (PR3) and myeloperoxidase (MPO) on monocyte subsets. There was no change in the proportion of monocytes in each subset in patients with AAV compared with healthy controls. The expression of CD14 on monocytes from patients with active AAV was increased, compared with patients in remission and healthy controls (P < 0·01). Patients with PR3‐ANCA disease in remission also had increased monocyte expression of CD14 compared with controls (P < 0·01); however, levels in patients with MPO‐ANCA disease in remission were lower than active MPO‐ANCA patients, and not significantly different from controls. There was a correlation between CD14 and both PR3 and MPO expression on classical monocytes in AAV patients (r = 0·79, P < 0·0001 and r = 0·42, P < 0·005, respectively). In conclusion, there was an increase in monocyte CD14 expression in active AAV and PR3‐ANCA disease in remission. The correlation of CD14 expression with ANCA autoantigen expression in AAV may reflect cell activation, and warrants further investigation into the potential for increased CD14 expression to trigger disease induction or relapse.     

Interference of PR3-ANCA with the enzymatic activity of PR3: differences in patients during active disease or remission of Wegener's granulomatosis

Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity. To investigate the relationship between functional effects of PR3-ANCA and disease activity, we tested the effect of IgG samples from sera of 43 WG patients, taken during active disease or remission, for their capacity to interfere with the proteolytic activity of PR3. Furthermore, longitudinal sera of seven WG patients were included. The enzymatic activity of PR3 was determined (1) with casein or with a small synthetic substrate and (2) by complexation of PR3 with alpha1-antitrypsin (alpha1-AT). With a fixed concentration (100 microg/ml) of IgG, PR3-ANCA from patients during an active phase of WG had a higher inhibitory capacity towards the proteolytic activity of PR3 and complexation of PR3 with alpha1-AT than did PR3-ANCA from WG patients during remission. However, the number of PR3-ANCA units that gave 50% inhibition of the PR3 enzymatic activity and its complexation with alpha1-AT was lower for patients during remission than for patients during an active phase of WG, indicating a stronger inhibitory capacity at a molar base. In conclusion, PR3-ANCA from patients during remission had a relatively higher inhibitory capacity towards the enzymatic activity of PR3 than PR3-ANCA from patients during an active phase. This may indicate that during active disease the ANCA titre is increased, but the number of active ANCA molecules that recognize the enzyme-inhibiting epitopes is not increased.     

Identification of MPO‐positive capillaries of the pleura by immunohistochemistry in MPO‐ANCA associated vasculitis

We present a case of a middle‐aged woman with myeloperoxidase anti‐neutrophil cytoplasmic autoantibody (MPO‐ANCA)‐associated vasculitis that demonstrated immunohistochemically positive MPO capillaries of the pleura. The patient initially presented with proteinuria and microscopic hematuria at the age of 38. Acute progressive glomerulonephritis and pulmonary hemorrhage occurred 4 years later, and a high serum titer of MPO‐ANCA was detected therefore a diagnosis of microscopic polyangiitis was made. Steroid‐pulse therapy was performed and the pulmonary shadow improved, but the renal failure did not improve, thus, hemodialysis was initiated. Thereafter, an 18‐year asymptomatic phase followed, but high serum levels of MPO‐ANCA persisted during this period. Chronic pulmonary hemorrhage was discovered at the age of 60, and video‐assisted thoracoscopic surgery was performed. Resected tissue revealed diffuse aloveolar hemorrhage accompanied by marked hemosiderin deposition, whereas MPO‐immunopositive capillaries were identified only in the pleura. To our knowledge, this is the first report demonstrating MPO‐positive capillaries in a disease other than glomerulonephritis. Judging from this unique case, MPO‐positive endothelial cells may appear only during the hyperacute stage before hemorrhage, and may diminish thereafter, thus, may be associated with the trigger of microscopic polyangiitis.     

Neutrophil extracellular trap formation is associated with autophagy‐related signalling in ANCA‐associated vasculitis

Increasing evidence indicates that aberrant neutrophil extracellular trap (NET) formation could contribute to the pathogenesis of anti‐neutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). Recent research has provided evidence that a novel type of ANCA autoantibody, anti‐lysosomal membrane protein‐2 (LAMP‐2) antibody, may have a pathogenic role in AAV. We have shown previously that anti‐LAMP‐2 antibody‐stimulated NET formation contains autoantigens and anti‐microbial peptides. The current study sought to determine whether LAMP‐2, as a novel antigen of ANCA, was present on NETs in AAV patients, the influence of the anti‐LAMP‐2 antibody on the neutrophil apoptosis rate and the role of autophagy in anti‐LAMP‐2 antibody‐induced NET formation. NET formation was assessed using immunofluorescence microscopy, scanning electron microscopy or live cell imaging. The neutrophil apoptosis rate was analysed using fluorescence activated cell sorting (FACS). Autophagy was detected using LC3B accumulation and transmission electron microscopy. The results showed that enhanced NET formation, which contains LAMP‐2, was observed in kidney biopsies and neutrophils from AAV patients. The apoptosis rate decreased significantly in human neutrophils stimulated with anti‐LAMP‐2 antibody, and this effect was attenuated by the inhibitors of autophagy 3‐methyladenine (3MA) and 2‐morpholin‐4‐yl‐8‐phenylchromen‐4‐one (LY294002). The anti‐LAMP‐2 antibody‐stimulated NET formation was unaffected by benzyloxycarbonyl‐Val‐ Ala‐Asp (OMe)‐fluoromethylketone (zVAD‐fmk) and necrostatin‐1 (Nec‐1), which are inhibitors of apoptosis and necrosis, respectively, but was inhibited by 3MA and LY294002. Moreover, the proportion of LC3BI that was converted to LC3BII increased significantly (P = 0·0057), and massive vacuolizations that exhibited characteristics typical of autophagy were detected in neutrophils stimulated with anti‐LAMP‐2 antibody. Our results provide further evidence that autophagy is involved in ANCA‐induced NET formation in human neutrophils.     

Longitudinal studies of patients with ANCA vasculitis demonstrate concurrent reactivity to complementary PR3 protein segments cPR3m and cPR3C and with no reactivity to cPR3N

Antibodies recognizing the complement of the middle of PR3 (cPR3m) occur in ～30% of PR3-anti-neutrophil cytoplasmic autoantibodies (ANCA)-vasculitis patients and immunization of animals with a peptide complementary to the middle of PR3 (cPR3m) induces not only anti-complementary PR3 antibodies, but also anti-PR3 antibodies derived through an anti-idiotypic response. PR3 epitopes recognized by patient ANCA, however, are not restricted to the middle of PR3. This prompted us to test for antibodies that react with proteins complementary to the terminal regions of PR3 (cPR3C and cPR3N) in PR3-ANCA patients. Anti-cPR3C reactivity was detected in 28% of patients but anti-cPR3N reactivity in only 15%. Ranked anti-cPR3C and anti-cPR3m reactivity correlated in the cohort, whereas there was no significant relationship between cPR3C and cPR3N reactivity. Serial samples from 3 patients' revealed that anti-cPR3C and anti-cPR3m reactivity followed a similar pattern over time. Serial samples from a fourth patient demonstrated an anti-cPR3N response without concurrent cPR3m or cPR3C reactivity. Epitope determination by mass spectrometry identified a 13-amino acid sequence on cPR3C that contained a common binding site recognized by antibodies from three patients. This peptide sequence contains a “PHQ” motif which was reported to be the basis for cross-reactivity of anti-cPR3m antibodies with plasminogen. Why these antibodies are detected in only ～30% of the patients remains unclear. The data reveal that it is not due to lack of inclusion of flanking regions of complementary PR3 during screening. Instead, quite unexpectedly, the data demonstrate that patients' antibodies react with a restricted epitope that exists in both cPR3m and cPR3C.     

Anti-endothelial cell antibodies (AECA) in patients with propylthiouracil (PTU)-induced ANCA positive vasculitis are associated with disease activity

Increasing evidence has demonstrated that propylthiouracil (PTU) could induce ANCA positive vasculitis. However, our previous work has suggested that only one-fifth of the PTU-induced ANCA positive patients had clinical vasculitis and so the mechanism is not clear. Anti-endothelial cell antibodies (AECA) have been implicated in the pathogenesis of various vasculitides, including primary ANCA positive systemic vasculitis. The purpose of this study is to investigate the prevalence of AECA and their possible role in the pathogenesis of patients with PTU-induced ANCA positive vasculitis. Sera from 11 patients with PTU-induced ANCA positive vasculitis at both active and quiescent phases, and sera from 10 patients with PTU-induced ANCA but without clinical vasculitis, were studied. Sera from 30 healthy blood donors were collected as normal controls. Soluble proteins from 1% Triton-100 extracted in vitro cultured human umbilical vein endothelial cells were used as antigens and an immunoblotting technique was performed to determine the presence of AECA, and their specific target antigens were identified. In patients with PTU-induced ANCA positive vasculitis, 10 of the 11 patients in an active phase of disease were serum IgG-AECA positive and six protein bands of endothelial antigens could be blotted (61 kD, 69 kD, 77 kD, 85 kD, 91 kD and 97 kD). However, in the quiescent phase, seven of the 10 positive sera turned negative. None of the ANCA positive but vasculitis negative patients or normal controls were AECA positive. In conclusion, AECA could be found in sera from patients with PTU-induced ANCA positive vasculitis and were associated more closely with vasculitic disease activity.     

Th17 and Treg lymphocytes as cellular biomarkers of disease activity in Granulomatosis with Polyangiitis

Granulomatosis with Polyangiitis (GPA) (formerly known as Wegener's granulomatosis) is a vasculitis of unknown etiology affecting predominantly small‐ to medium‐sized vessels, usually involving the upper and lower respiratory tract and kidneys. Anti‐neutrophil cytoplasmic autoantibodies are probably the initial cause of the inflammatory process that leads to the typical necrotizing lesions. In this issue of the European Journal of Immunology, Szczeklik et al. [Eur. J. Immunol. 2017. 47: 724–733] report some interesting findings on the possible involvement of T‐cell subsets in the pathogenesis of the disease. This prospective study, performed on a large cohort of patients, identifies Th17 lymphocytes as the possible pathogenic subset of GPA, and Treg cells as the possible suppressors of the inflammatory process. These two subsets in peripheral blood could be used as cellular biomarkers of disease activity, and this would result particularly useful in the follow‐up of patients once the immunosuppressive treatment has been initiated.     

Alpha 1-antitrypsin deficiency and anti-proteinase 3 antibodies in anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis.

alpha 1-antitrypsin (alpha 1-AT) is a naturally occurring inhibitor of proteinase 3 (PR3) and elastase, two of the target antigens of anti-neutrophil cytoplasmic antibodies (ANCA). An increased incidence of alpha 1-AT phenotypes associated with dysfunctional alpha 1-AT or low serum levels has been reported in patients with anti-PR3 antibodies. We have studied the relationship between ANCA, and phenotypes and serum levels of alpha 1-AT. Phenotypes usually associated with a moderate or severe reduction in alpha 1-AT serum levels or in dysfunctional activity were found more often in individuals with anti-PR3 antibodies than in the general population: four of the 31 patients (13%) with anti-PR3 antibodies had phenotypes MZ (n = 2), S (n = 1) or Z (n = 1) (P < 0.05). However, the corresponding alpha 1-AT serum levels were normal (n = 3) or elevated (n = 1). None of the 31 sera with anti-PR3 antibodies had low levels of alpha 1-AT. No abnormal alpha 1-AT phenotype was demonstrated in seven patients with anti-elastase antibodies, despite a low level of alpha 1-AT in one serum. Anti-myeloperoxidase antibodies are common in patients with ANCA, but no abnormal phenotype or low serum alpha 1-AT level was demonstrated in any of 29 sera containing these antibodies. Finally anti-glomerular basement membrane (GBM) antibodies occur occasionally in patients with ANCA-associated diseases, but again none of 10 sera had an abnormal alpha 1-AT phenotype or low serum level. ANCA were not demonstrated by indirect immunofluorescence in any serum from 73 patients with abnormal alpha 1-AT phenotypes. These results confirm that patients with anti-PR3 antibodies often have alpha 1-AT phenotypes that are usually associated with low serum levels of alpha 1-AT or with dysfunctional protein. Nevertheless, the incidence of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is very low. This probably reflects the rarity of Wegener's granulomatosis, the major disease associated with anti-PR3 antibodies, and the relative frequency of abnormal alpha 1-AT phenotypes. The mechanism for the development of anti-PR3 antibodies in patients with abnormal alpha 1-AT phenotypes is not clear, but may relate to the increased propensity of unbound and uninhibited PR3 to stimulate autoantibody production.     

Intestinal anti‐tissue transglutaminase antibodies in potential coeliac disease

Anti‐tissue transglutaminase 2 (anti‐TG2) antibodies are present in the serum of the great majority of untreated coeliac disease (CD) patients. They are produced and deposited in the small intestinal mucosa. Potential CD patients present serum anti‐TG2 antibodies higher than cut‐off, but a normal duodenal mucosa where mucosal deposits of anti‐TG2 are not always detectable. The aim of our work was to investigate the presence of anti‐TG2 intestinal antibodies in patients with potential CD, and identify the most sensitive test to detect them. Twelve active CD patients, 28 potential CD patients and 39 non‐CD controls were enrolled. Biopsy fragments from all patients were analysed by double immunofluorescence to detect mucosal deposits of anti‐TG2 antibodies. Fragments from the same subjects were also cultured for 24 h with medium in the presence or absence of gliadin peptides. Anti‐TG2 autoantibodies secreted into supernatants were measured by enzyme‐linked immunosorbent assay. All active CD, 68% of potential CD patients and 20% of non‐CD controls showed mucosal deposits of immunoglobulin (Ig)A anti‐TG2; at the same time 100, 96 and 8% of active CD, potential CD and non‐CD control patients secreted these antibodies in culture supernatants, respectively. Our data showed that, to detect intestinal anti‐TG2 antibodies, the measurement of antibodies secreted into culture supernatants has higher sensitivity and specificity (97·5 and 92·3%, respectively) than the detection of mucosal deposits (77·5 and 80·0%, respectively). The measurement of intestinal anti‐TG2 antibodies may prove useful in clinical practice to predict evolution towards mucosal atrophy in potential coeliac patients and identify patients with gluten sensitivity.     

Interferon‐γ enhances both the anti‐bacterial and the pro‐inflammatory response of human mast cells to Staphylococcus aureus

Human mast cells (huMCs) are involved in both innate and adaptive immune responses where they release mediators including amines, reactive oxygen species (ROS), eicosanoids and cytokines. We have reported that interferon‐γ (IFN‐γ) enhances FcγR‐dependent ROS production. The aim of this study was to extend these observations by investigating the effect of IFN‐γ on the biological responses of huMCs to Staphylococcus aureus. We found that exposure of huMCs to S. aureus generated intracellular and extracellular ROS, which were enhanced in the presence of IFN‐γ. IFN‐γ also promoted bacteria killing, β‐hexosaminidase release and eicosanoid production. Interferon‐γ similarly increased expression of mRNAs encoding CCL1 to CCL4, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), tumour necrosis factor‐α and CXCL8 in S. aureus‐stimulated huMCs. The ability of IFN‐γ to increase CXCL8 and GM‐CSF protein levels was confirmed by ELISA. Fibronectin or a β₁ integrin blocking antibody completely abrogated IFN‐γ‐dependent S. aureus binding and reduced S. aureus‐dependent CXCL8 secretion. These data demonstrate that IFN‐γ primes huMCs for enhanced anti‐bacterial and pro‐inflammatory responses to S. aureus, partially mediated by β₁ integrin.     

IFN‐λ‐mediated IL‐12 production in macrophages induces IFN‐γ production in human NK cells

With increasing interest in alternative options to interferon‐alpha‐based treatments, IFN‐λ has shown therapeutic promise in a variety of diseases. Although the antiviral activity of IFN‐λ has been extensively studied, there is limited knowledge regarding the immunological functions of IFN‐λ and how these differ from those of other classes of IFNs. In this study, we investigated the effects of IFN‐λ on primary human NK cells, both in a direct and indirect capacity. We demonstrate that in contrast to interferon‐alpha, IFN‐λ is unable to directly stimulate NK cells, due to the absence of IFN‐λ receptor chain 1 (IFN‐λR1) on NK cells. However, IFN‐λ, in combination with TLR4 challenge, is able to induce the production of select members of the IL‐12 family of cytokines in monocyte‐derived macrophages. We further show that through macrophage‐mediated IL‐12 production, IFN‐λ is able to indirectly affect NK cells and ultimately induce IFN‐γ production.     

Antigen‐triggered interferon‐γ and interleukin‐10 pattern in cured mucosal leishmaniasis patients is shaped during the active phase of disease

An exacerbated type 1 response to leishmanial antigens is the basis of tissue destruction observed in mucosal leishmaniasis (ML). After therapy, a persistent production of high levels of inflammatory cytokines can confer a poor prognosis. Herein we investigated whether the clinical conditions defined during the active phase of ML affect the magnitude of long-term anti-Leishmania immune response. Twenty clinically cured ML cases were studied. Peripheral blood mononuclear cells (PBMC) were cultured with L. braziliensis antigens (Lb-Ag), Toxoplasma gondii antigens (Tg-Ag), concanavalin-A (Con-A) or medium alone, and the lymphocyte proliferative response and cytokine secretion were quantified. Medical records were reviewed for Montenegro skin test (MST) during diagnosis, duration of ML disease or time elapsed after clinical cure. The duration of disease was correlated positively with MST (r = 0·61). Lb-Ag induced interferon (IFN)-γ was correlated positively with duration of illness (r = 0·69) as well as the frequency of secreting cells [enzyme-linked immunospot (ELISPOT)] assay. No association was observed for Tg-Ag or Con-A. Disease duration was correlated negatively with interleukin (IL)-10 production (r = −0·76). Moreover, a negative correlation between length of time after clinical cure and TNF levels (r = −0·94) or the IFN-γ : IL-10 ratio (r = −0·89) were also seen. We suggest that the magnitude of the IFN-γ inflammatory response triggered by ML can be driven by the time of leishmanial antigens exposition during the active phase of the disease. This pattern could persist even long-term after cure. However, despite IFN-γ levels, the decrease of the TNF and IFN-γ : IL-10 ratio reflects the control of proinflammatory responses achieved by cure of ML, possibly preventing disease relapses.     

Immunoglobulin (Ig)M antibodies to proteinase 3 in granulomatosis with polyangiitis and microscopic polyangiitis

Anti‐neutrophil cytoplasmic antibodies (ANCA) appear to play an important role in the pathogenesis of ANCA‐associated vasculitis (AAV). However, ANCA alone are not sufficient to generate disease, and some evidence suggests that infectious triggers may serve as inciting events for AAV disease activity. Antibodies of the immunoglobulin (Ig)M isotype often serve as markers of recent infection, and IgM ANCA have been identified previously in patients with AAV, although the frequency and clinical relevance of IgM ANCA is not well established. We sought to characterize IgM ANCA more clearly by creating a novel enzyme‐linked immunosorbent assay (ELISA) for IgM antibodies to proteinase 3 [IgM proteinase 3 (PR3)–ANCA], which we applied to two large, clinically well‐characterized trial cohorts of patients with granulomatosis with polyangiitis and microscopic polyangiitis. In the first cohort, IgM PR3–ANCA occurred with a frequency of 15·0%, and were associated with a higher degree of disease severity and a trend towards a higher rate of alveolar haemorrhage (29·6 versus 15·7%, P = 0·10). Analysis of follow‐up samples in this cohort showed that the presence of IgM PR3–ANCA was transient, but could recur. In the second cohort, IgM PR3–ANCA occurred with a frequency of 41·1%, and were also associated with a higher degree of disease severity. A higher rate of alveolar haemorrhage was observed among those with IgM PR3–ANCA (45·3 versus 15·8%; P < 0·001). The association of transient IgM PR3–ANCA with an acute respiratory manifestation of AAV suggests a possible link between an infectious trigger and AAV disease activity.     

Card9‐dependent IL‐1β regulates IL‐22 production from group 3 innate lymphoid cells and promotes colitis‐associated cancer

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell‐specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL‐1β production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis‐associated cancer (CAC) model. Card9^?/? mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL‐1β generation and defective IL‐1β controlled IL‐22 production from group 3 innate lymphoid cells. Consistent with the key role of immune‐derived IL‐22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9^?/? mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9‐controlled, ILC3‐mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9‐mediated innate immunity in inflammation‐associated carcinogenesis.