Recognition and control of neutrophil extracellular trap formation by MICL

Regulation of neutrophil activation is critical for disease control. Neutrophil extracellular traps (NETs), which are web-like structures composed of DNA and neutrophil-derived proteins, are formed following pro-inflammatory signals; however, if this process is uncontrolled, NETs contribute to disease pathogenesis, exacerbating inflammation and host tissue damage 1,2 . Here we show that myeloid inhibitory C-type lectin-like (MICL), an inhibitory C-type lectin receptor, directly recognizes DNA in NETs; this interaction is vital to regulate neutrophil activation. Loss or inhibition of MICL functionality leads to uncontrolled NET formation through the ROS–PAD4 pathway and the development of an auto-inflammatory feedback loop. We show that in the context of rheumatoid arthritis, such dysregulation leads to exacerbated pathology in both mouse models and in human patients, where autoantibodies


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via two NET proteins: LL-37, which potentiates a type I IFN induction, and HMGB1, which acts through the receptor for advanced glycation products (RAGE) [18][19][20][21][22] .Recently, it has been shown that TLR4 recognizes and is activated by NET-associated histones, whereas histone containing chromatin DNA regulates TLR4 recruitment to endosomes 23 .However, the mechanisms that regulate cellular responses to NETs are still incompletely understood 23 .
Loss or mutation of inhibitory receptors are often associated with unchecked inflammation and destructive autoimmunity 24 .Recent work from our group has indicated that MICL (also known as CLEC12A) is one such receptor, which can regulate the pathogenesis of rheumatoid arthritis 25 .In contrast to wild-type (WT) mice, we showed that MICL-deficient animals (Micl −/− ) undergoing collagen antibody-induced arthritis (CAIA) presented with exacerbated joint inflammation that did not resolve 25 .Through investigating the underlying mechanism, we discovered that MICL functions as an essential pattern recognition receptor (PRR) for NETs on neutrophils.Recognition of NETs by MICL inhibits neutrophil activation and further NET formation, regulating a positive-feedback cycle that, on the one hand, protects from excessive inflammation during multiple autoimmune diseases, but on the other hand, increases susceptibility to invasive infections.

MICL regulates neutrophil responses
Micl −/− mice exhibit enhanced and non-resolving joint inflammation compared with WT mice during CAIA 25 (Extended Data Fig. 1a).To understand the underlying mechanism, we first determined the cell type (or types) responsible for this phenotype by analysing the cellular infiltrate in the hind paws of arthritic mice by flow cytometry.Day 7 was selected for analysis, representing the peak clinical score in WT mice undergoing CAIA (Extended Data Fig. 1a).We found a significant increase in neutrophils (defined as CD45 + Ly6G + CD11b + cells) in the joints of Micl −/− animals, whereas other myeloid populations were unaffected (Fig. 1a,b and Extended Data Fig. 1b).Analyses of an earlier time point (day 5) showed that neutrophil numbers were higher in the joints of MICL-deficient mice even during early stages of inflammation, when there was no apparent clinical difference between the two groups of mice, again without affecting other myeloid populations (Extended Data Fig. 1c).Of note, MICL deficiency also exacerbated joint inflammation in another effector phase model of rheumatoid arthritis: the K/BxN serum transfer model 26 (Extended Data Fig. 2a).As in CAIA, Micl −/− mice in the K/BxN serum transfer model presented higher numbers of neutrophils than WT mice (Extended Data Fig. 2b).There were no alterations in the frequency of cellular populations in the bone marrow or blood of naive Micl −/− mice (Extended Data Fig. 2c,d).
We next analysed the expression of key neutrophil adhesion and activation molecules (CD18, CD11b and CD62L) and chemotactic receptors (CXCR2 (ref.27), CCR1 and C5aR 28,29 ) by flow cytometry.Neutrophils in the joints of Micl −/− mice under CAIA exhibited increased expression of CD18 and CD11b, and decreased expression of the lymphoid organ homing receptor CD62L, compared with WT animals, indicative of a heightened state of activation (Extended Data Fig. 2e).This activated phenotype was only observed in cells isolated from the joints and not on neutrophils concomitantly isolated from the peripheral blood of mice undergoing CAIA (Extended Data Fig. 2e).Analysis of receptors controlling neutrophil migration revealed increased expression of C5aR and the chemokine receptor CXCR2 on neutrophils isolated from the joints of Micl −/− mice, compared with WT animals, although the increase of the former was not statistically significant (Extended Data Fig. 2f).By contrast, expression levels of CXCR2 and C5aR were comparable between neutrophils isolated from the peripheral blood of WT and Micl −/− mice (Extended Data Fig. 2f).The expression of CCR1 on neutrophils was unaffected by MICL deficiency (Extended Data Fig. 2f).MICL-deficient animals in the K/BxN serum transfer model of arthritis showed similar changes in neutrophil activation (Extended Data Fig. 2g).There were no differences in the expression of key adhesion and activation molecules and chemotactic receptors in the bone marrow or blood neutrophils of naive animals (Extended Data Fig. 2h,j).Together, these data demonstrate that MICL deficiency is characterized by an increased number and an enhanced activation of neutrophils in the joints of mice during CAIA and in the K/BxN serum transfer model of arthritis.
Neutrophils contribute significantly to the pathology of CAIA 30 .To investigate whether these cells were solely responsible for the exacerbated disease in Micl −/− mice, we successfully depleted circulating neutrophils via anti-Ly6G administration following the onset of CAIA (Fig. 1c,d and Extended Data Fig. 3a).Of note, clinical disease was significantly reduced following neutrophil depletion, and to an equivalent level, in both WT and Micl −/− mice (Fig. 2e).A similar reduction in disease severity occurred in both groups of mice following neutrophil depletion using the less-specific marker GR-1 (Extended Data Fig. 3b-e).These data show that the elevated pathology in the Micl −/− mice undergoing CAIA was stemming from the neutrophil compartment.
We then investigated whether the aberrant neutrophil response in the Micl −/− mice resulted from a cell-intrinsic or cell-extrinsic defect.For this, we isolated neutrophils from the bone marrow of naive WT or Micl −/− mice (Extended Data Fig. 3f) and performed a single adoptive transfer of these cells into arthritic WT mice at day 7 post-induction of CAIA (Fig. 1f).We detected transferred cells in the joints even 48 h post-transfer (Extended Data Fig. 3g).Although transfer of WT neutrophils did not significantly alter disease severity (Fig. 1g,h), transfer of MICL-deficient neutrophils into WT mice induced a significant increase in joint inflammation, determined by both clinical score and histological changes (Fig. 1g,h).Together, these data reveal that aberrant neutrophil function underlies the pathology of Micl −/− mice during CAIA.

MICL controls NET formation
We then determined how loss of MICL was affecting neutrophil function.A previous report has suggested that MICL negatively regulates the respiratory burst in neutrophils in response to specific ligands, such as uric acid crystals (monosodium urate (MSU)) 31 .Indeed, we recapitulated this observation and showed that Micl −/− neutrophils induced a significantly elevated ROS in response to MSU, but not to phorbol-12-myristate-13-acetate (PMA) or zymosan, a potent inducer of ROS and a ligand for a related C-type lectin (Dectin-1) 32 , respectively (Fig. 2a).Micl −/− neutrophils also produced higher levels of ROS in response to A. fumigatus hyphae, which are also known to induce ROS 33 (Fig. 2a).As ROS production is involved in induction of NETs 34 , we examined this programmed cell death response and found that neutrophils from Micl −/− mice exhibited significantly increased NET formation in vitro following MSU stimulation (Fig. 2b and Extended Data Fig. 4,b).NET formation was unaltered following PMA stimulation (Fig. 2b and Extended Data Fig. 4a).The ability of diphenyleneiodonium chloride (DPI) to inhibit NET formation in cells from MICL-deficient animals revealed the dependence of this response on NADPH oxidase (Extended Data Fig. 4c).MICL transduces intracellular inhibitory signals through SHP1/2 (refs.35,36); we found that inhibition of SHP1/2 increased NET formation in WT neutrophils, but not in MICL-deficient neutrophils, indicating that inhibitory signalling from MICL was required to regulate this response (Extended Data Fig. 4d).Of note, we detected increased NET formation (defined as GR-1 + cit-H3 + DNA/H1 + -stained cells) in the joints of Micl −/− mice undergoing CAIA at day 11 by immunofluorescence (Fig. 2c and quantification in Extended Data Fig. 4e) and by imaging flow cytometry (Fig. 2e and Extended Data Fig. 4f).
In addition to ROS, the formation of NETs can require histone citrullination through protein arginine deaminase 4 (PAD4) 37 .Using two different PAD4 inhibitors, BB-CL-amidine 38 and GSK484 (ref.39), complete inhibition of NET formation in vitro was shown (Extended Data Fig. 5a).In vivo (Fig. 2d), imaging flow cytometry revealed that treatment with these inhibitors led to a significant reduction in the number of NETs detectable in the joints of Micl −/− but not WT mice during CAIA (Fig. 2e).There was no effect of the inhibitor on the number of neutrophils recruited into the joints of these mice (Fig. 2f).Treatment of Micl −/− mice with both PAD4 inhibitors reduced the severity of the disease back to WT levels (Fig. 2g and Extended Data Fig. 5b).There was no significant effect of the inhibitors on disease development in WT mice (Fig. 2g and Extended Data Fig. 5b), as expected 40 .Specific PAD4 inhibition also reduced disease severity in MICL-deficient mice during the K/BxN serum transfer model of arthritis (Extended Data Fig. 5c).Administration of DNase I to degrade NETs in vivo ameliorated the enhanced clinical severity in the Micl −/− mice undergoing CAIA, but had no effect on disease severity in WT mice (Extended Data Fig. 5d).Thus, these data show that aberrant regulation of NET formation underlies the pathology of MICL-deficient mice during models of arthritis.

Anti-MICL antibodies link to NET diseases
We had previously shown that administration of antibodies targeting MICL was able to exacerbate CAIA in WT mice, recapitulating the phenotype of MICL-deficient animals 25 .Here we found that treatment of WT neutrophils with anti-MICL antibodies resulted in elevated ROS production in response to MSU, but not zymosan, suggesting that these antibodies were blocking receptor functionality (Extended Data Fig. 6a).To gain more insight into the effect of anti-MICL antibodies on neutrophil activation, we administered anti-MICL monoclonal antibodies to WT mice during CAIA in the presence of NET inhibitors.As previously observed, administration of anti-MICL antibodies exacerbated CAIA pathology in WT mice (Extended Data Fig. 6b).Administration of a PAD4 inhibitor completely reduced disease symptoms to the levels seen in the isotype control-treated mice (Extended Data Fig. 6b), revealing that aberrant NET formation was underlying the effect of the anti-MICL antibodies on disease pathology.
To further substantiate the receptor-blocking effect of anti-MICL antibodies, we tested them using an alternative model: collageninduced arthritis (CIA).This is an important T cell-dependent model Article of arthritis that recapitulates both the pathophysiology and the histological presentation of human disease, although C57BL/6 mice, the same background on which our knockout model was based, are resistant to this model of disease 41 .However, the ability of anti-MICL antibodies to alter MICL function enabled us to explore the role of this receptor during CIA induced in DBA/1 mice.As before, administration of anti-MICL antibodies to DBA/1 mice exacerbated CIA disease and increased the number of neutrophils in the joints (Extended Data Fig. 6c).These results confirm that antibodies to MICL interfere with key functions of this receptor and its ability to regulate neutrophil activation, exacerbating arthritic inflammation in different mouse models.
As we previously detected the presence of anti-MICL antibodies in a small cohort of patients with rheumatoid arthritis 25 , we explored whether these antibodies could affect neutrophil activation.Here we found that anti-human MICL antibodies were able to enhance the respiratory burst and NET formation in human neutrophils in vitro in response to MSU, but not to PMA (Fig. 3a,b).Moreover, serum samples from patients with rheumatoid arthritis were able to increase ROS production in human neutrophils following stimulation with MSU (Fig. 3c and Extended Data Fig. 6d); this effect directly correlated with the anti-MICL serum titres, demonstrating that patient anti-MICL antibodies interfere with MICL function.
To determine whether anti-MICL antibodies were influencing clinical severity, we further analysed the prevalence of these antibodies in patients with rheumatoid arthritis, using an ELISA-based assay 25 .For this, we interrogated 200 serum samples from patients, along with matched controls, obtained from the Scottish Early Rheumatoid Arthritis (SERA) cohort 42 .We found that the majority of patients with rheumatoid arthritis presented with significantly elevated titres of anti-MICL autoantibodies (Fig. 3d).High levels of autoantibodies were also detected in some healthy controls without rheumatoid arthritis.Although we did not find an association between the level of anti-MICL autoantibodies and DAS28 or Sharp erosion scores (data not shown), after correcting for age and sex, we detected an association between the level of anti-MICL antibodies and the level of rheumatoid factor, an autoantibody targeting the Fc region of IgG antibodies, which is used to diagnose, classify and predict development of rheumatoid arthritis 43,44 (Fig. 3e).We also found a significant correlation between the level of anti-MICL autoantibodies and the level of anti-cyclic citrullinated peptide (CCP) antibodies in patients with rheumatoid arthritis (Fig. 3f), indicative of a direct link of serum anti-MICL antibodies with NETosis and disease severity 45,46 .Together, these results show that autoantibodies targeting MICL interfere with the function of this receptor and correlate with severity of rheumatoid arthritis in patients.
We next determined whether anti-MICL autoantibodies were associated with any other NET-linked inflammatory diseases.Of note, we found high titres of anti-MICL antibodies in patients with SLE (Fig. 3g) and severe COVID-19 (Fig. 3h), both of which are inflammatory disorders where disease severity has been linked to NETosis 15,47 .Moreover, as we found in rheumatoid arthritis, serum samples containing anti-MICL antibodies from patients with SLE or severe COVID-19 were able to modulate neutrophil function, as demonstrated by increased ROS production following MSU stimulation (Extended Data Fig. 6e,f).We demonstrated the specificity of this response through the addition of a soluble chimeric protein containing the C-type lectin-like domain (CTLD) of human MICL, which abrogated the serum effect on the neutrophil response (Fig. 3i and Extended Data Fig. 6g).A related control CTLD had no effect on these responses (Extended Data Fig. 6g).Thus, autoantibodies that modulate MICL-mediated neutrophil functions are present in patients with a wide variety of NET-related pathologies.

MICL is a PRR for NETs
Given that NETs can activate pro-inflammatory functions of naive neutrophils 48 , we wondered whether antibody-mediated interference or loss of MICL function was affecting neutrophil responses to NETs themselves.We observed that following exposure to preformed NETs, MICL-deficient neutrophils induced significantly increased levels of ROS and higher formation of NETs compared with the response of WT neutrophils (Fig. 4a,b and Extended Data Fig. 7a,b).ROS production in MICL-deficient cells was unaltered when neutrophils were stimulated with NETs treated with polymyxin B (Extended Data Fig. 7c).Following stimulation of cells from both groups of mice with preformed NETs, the ability of both ROS (DPI) and NET (PAD4) inhibitors to prevent NET formation shows that this process is dependent on NADPH and

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PAD4 activity (Extended Data Fig. 7d).To show that human MICL was functioning similarly, we generated MICL-knockout human neutrophils derived from CD34 + haematopoietic progenitors 49 (Extended Data Fig. 7e) and found increased ROS production in response to preformed NETs compared with control cells (Extended Data Fig. 7f).
Of note, we also demonstrated that normal neutrophils isolated from healthy human controls had significantly elevated ROS responses to human NETs in the presence of anti-human MICL antibodies (Fig. 4c).
To determine whether MICL was functioning as a PRR for NETs themselves, we examined the ability of a soluble chimeric protein consisting of the CTLD of mouse MICL fused to the Fc region of human IgG1 (Fc-mMICL) to directly recognize NETs.Using this Fc-protein and a structurally related control protein, we showed that MICL binds directly to NETs using an ELISA-based assay (Fig. 4d).Moreover, recognition of NETs by MICL could also be demonstrated in a cellular context using MICL-expressing reporter cells 50 (Fig. 4e).MICL recognition of NETs was blocked in the presence of antibodies targeting MICL (Extended Data Fig. 7g), showing that antibodies to MICL block the ability of the receptor to recognize its ligand.To determine which component of NETs is required to interact with this receptor, we treated NETs with DNase I or proteinase K, and found that treatment with DNase abolished the ability of MICL to recognize NETs (Fig. 4d,e).Furthermore, MICL-deficient neutrophils stimulated with DNase-treated NETs induced significantly lower levels of ROS (Extended Data Fig. 7h).Consistent with these observations, MICL was able to recognize genomic DNA, and this interaction could be blocked in the presence of anti-MICL antibodies (Extended Data Fig. 7i).
To show a direct role for MICL in response to NETs in vivo, we injected preformed NETs into the peritoneum of Micl −/− and WT mice.Neutrophil recruitment was significantly higher in Micl −/− mice treated with NETs, but similar to WT in mice administered with LPS (Fig. 4f), revealing a specific negative regulatory function of MICL following recognition of NETs.Moreover, there was a trend to higher levels of cell-free DNA measurements following NET administration in MICL-deficient mice (Extended Data Fig. 7j).Collectively, our data show that MICL functions as a PRR for NETs, regulating activation and NET formation in both mouse and human neutrophils.

MICL regulates fungal-induced NETs
To validate our hypothesis that NET sensing by MICL functions as a universal regulator of neutrophil activation and NET formation, we examined neutrophil responses elicited by A. fumigatus.NET formation is critical in the immune response to A. fumigatus, restraining fungal growth and preventing tissue dissemination 51,52 .We demonstrated that NETs are released in response to A. fumigatus hyphae 53,54 (Fig. 5a), and both MICL-deficient neutrophils and human neutrophils treated with anti-MICL antibodies induce higher levels of NET formation in vitro in response to this pathogen (Fig. 5a and Extended Data Fig. 8a).Of note, MICL-deficient mice were significantly more protected than WT mice following intravenous infection with A. fumigatus conidia (Fig. 5b).This increased susceptibility of WT mice was associated with increased fungal burdens in the brains of these animals at day 2 post-infection (Fig. 5c and Extended Data Fig. 8b).Analysis of inflammatory responses showed that serum levels of IL-6 and brain levels of G-CSF were significantly higher in infected WT than in MICL-deficient mice (Fig. 5d,e), showing that MICL functions to restrain systemic inflammation during invasive aspergillosis.Furthermore, we confirmed that the increased survival of MICL-deficient mice was associated with higher NET formation, as the treatment of these animals with a PAD4 inhibitor (GSK484), but not with the vehicle, increased the susceptibility of the knockout mice to A. fumigatus infection to levels similar to those observed in WT mice (Fig. 5f and vehicle control shown in Extended Data Fig. 8c).Moreover, administration of the inhibitor increased fungal burdens in the brains of MICL-deficient mice (Fig. 5g).There was no effect of the  NET inhibitor or vehicle control on disease development and fungal burdens in WT mice 53 (Fig. 5g and Extended Data Fig. 8c).Thus, dysregulation of NET formation in MICL-deficient mice results in increased resistance to a systemic fungal infection.

Discussion
The integration of signalling between diverse receptors helps to refine and modulate immune responses 55 .Inhibitory receptors regulate immune cell activation, avoiding excessive inflammation and host tissue damage.Our data revealed that MICL, an inhibitory C-type lectin receptor, is vital for the regulation of key neutrophil functions.We discovered that MICL is a PRR for NETs, and that this interaction is essential for controlling key neutrophil responses, including the respiratory burst and further induction of NETs themselves.This regulation is critical for limiting NET-mediated inflammatory conditions, such as in arthritis, SLE or severe COVID-19 infections, in which MICL prevents the formation of a positive-feedback loop that leads to uncontrolled inflammation (Extended Data Fig. 9).By contrast, this regulation of NET formation supresses robust control of invasive infections, promoting disease susceptibility.
In the context of arthritis, we showed that loss of MICL functionality increases NET formation and neutrophil activation by NETs, triggering an inflammatory chain reaction that leads to increased joint inflammation in three mouse models of arthritis.We also found that anti-MICL antibodies block the receptor function, and that the presence of these antibodies influences the severity of disease in mouse models and in patients with rheumatoid arthritis.In fact, we demonstrated that serum samples from patients with rheumatoid arthritis possessing anti-MICL autoantibodies, and antibodies to mouse or human MICL, are able to recapitulate MICL-deficient neutrophils, in terms of dysregulation of ROS production and NET release.NETs are released locally in the inflamed joints of patients with rheumatoid arthritis, and the citrullinated neo-epitopes generated during this process promote the formation of anti-CCP antibody that contribute to the perpetuation of the disease 6,56 .Of note, we found that the level of autoantibodies to MICL correlate with the level of anti-CCP antibodies in patients, indicating a direct link between MICL function and NET formation in patients with rheumatoid arthritis.Moreover, we showed that autoantibodies capable of blocking MICL function are also present in other autoinflammatory conditions, including SLE and severe COVID-19, in which NETs are linked to disease pathology 15,47 .Thus, MICL represents a universal novel autoregulatory pathway that prevents aberrant neutrophil activation and subsequent tissue damage in autoimmunity.
Neutrophils also have a crucial role in protection against fungal pathogens, such as A. fumigatus 57 .Protective neutrophil responses to A. fumigatus hyphae involve ROS production and NET formation 51,52,54 .Although PAD4 is required for NET formation in response to A. fumigatus, previous studies using Pad4-knockout mice have suggested no role for this enzyme during infection 53 .Indeed, we found that PAD4 inhibition does not affect disease severity in WT mice.Here we discovered that the PAD4 pathway is stringently regulated by MICL, but that mice lacking this receptor use this pathway to restrict fungal infection through increased NET formation.Pharmacological inhibition of NET formation using a PAD4 inhibitor blocked this pathway in MICL-deficient mice, reverting their susceptibility to infection back to WT levels.Our data show that similar MICL-mediated control of the PAD4 pathway also occurs during autoimmune disease.Indeed, the PAD4 pathway has previously been shown not to be required for effector phase responses during arthritis 58 , as we found in our WT mice (Fig. 2g and Extended Data Fig. 5b).Of note, our data show that in the absence of MICL (or inhibition of receptor function by anti-MICL antibodies), the PAD4 pathway becomes activated during autoimmune disease, leading to dysregulated NET formation and aberrant inflammation.

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In conclusion, we defined a key mechanism by which MICL regulates neutrophil activation and NET formation.MICL directly recognizes NETs, inducing intracellular signalling that dampens neutrophil responses and preventing the formation of a positive-feedback loop leading to further NET formation.In patients with rheumatoid arthritis, SLE and severe COVID-19, this feedback loop becomes dysregulated by autoantibodies to MICL, leading to a worsened disease outcome.It is likely that autoantibodies to MICL are also associated with the severity of other NET-mediated autoimmune disorders, such as anti-neutrophil cytoplasmic antibody-associated vasculitis.Given that we also observed elevated levels of anti-MICL antibodies in a proportion of healthy individuals, future work should explore the possibility that the presence of such autoantibodies are associated with, or predispose towards, the development of autoimmune disease.Our data also reveal that NET sensing by MICL can have different clinical outcomes depending on the context.In this sense, our results suggest therapeutic targeting of MICL with antibodies to increase ROS and NET formation, contributing to fungal clearance, could be used to treat disseminated forms of invasive infection.Conversely, blocking of the antibody-receptor interaction could be of therapeutic benefit for NET-mediated inflammatory disease.In summary, our observations revealed a novel and fundamental function of inhibitory pathways underlying infectious and non-infectious disease pathogenesis.

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Mice
Micl −/− and C57BL/6J mice were bred and maintained under specific pathogen-free (SPF) conditions at the University of Aberdeen, University of Exeter and Charles River Laboratories.Mice were housed in separate groups with bedding exchanges between cages every 2 days for 1 week before commencement of experiments, and maintained on a 12 h-12 h dark-light cycle (07:00-19:00) at 20-24 °C and relative humidity of 55 ± 15%.Mouse experiments were performed by random assignation of age-matched (6-8 weeks old) and sex-matched mice in experimental or control groups at the beginning of each experiment; females were co-housed and experiments were not blinded.All experiments conformed to the ethical review committee of the University of Aberdeen, University of Exeter and the UK Home Office regulations (project license numbers: P79B6F297 and P6A6F95B5).

CAIA
Male mice received intraperitoneal (i.p.) injections of 2 mg of Arthri-toMab monoclonal antibody cocktail (MD Bioproducts) on day 0, followed by 50 µg of lipopolysaccharide (LPS) i.p. (MD Biosciences) on day 3. Joint inflammation was scored visually using a scale of 0 (no visible signs of redness or swelling) to 4 (extensive swelling with signs of deformity).To achieve neutrophil depletion during CAIA, 500 µg of rat anti-mouse Ly6G (clone 1A8, Bio-X-Cell) or isotype control (rat anti-mouse IgG2a; Bio-X-Cell) was administered i.p. to mice every 48 h from day 5 onwards.Alternatively, 50 µg of rat anti-mouse GR-1 (clone RB6-8C5) or isotype control (rat anti-mouse IgG2b) was administered i.p. every 48 h from day 5 onwards.Mice were culled on day 5, 7, 10, 11 or 13, as indicated in the text.For adoptive transfer experiments, bone marrow neutrophils were isolated as detailed below and stained with cell proliferation dye eFluor 670 (eBioscience).Labelled cells (5 × 10 6 ) were transferred intravenously (i.v.) to WT mice on day 7 following induction of CAIA.Mice were culled on day 9.To investigate the role of NET formation, mice undergoing CAIA were injected i.p. with 2 mg kg −1 daily of BB-CL-amidine (Cayman Chemical), 4 mg kg −1 daily of GSK484 (Cambridge Biosciences), 75 U per animal of DNase I (Merck) or vehicle (5% DMSO in 10% cyclodextran for BB-Cl-amidine or ethanol 99.9% diluted 1:50 in 0.9% NaCl for GSK484) from day 7 to day 10.Mice were culled on day 11.

K/BxN serum transfer model of arthritis
Male mice were administered i.p. with 100 µl of serum from transgenic K/BxN mice 26 .The development of clinical symptoms were monitored daily and mice were culled on day 10.Joint inflammation was scored visually using a scale of 0 (no visible signs of redness or swelling) to 4 (extensive swelling with signs of deformity).To investigate the role of NET formation, mice were injected i.p. with 4 mg kg −1 daily of GSK484 or vehicle (ethanol 99.9% diluted 1:50 in 0.9 % NaCl) from day 7 to day 10.Mice were culled on day 11.

CIA
Male DBA/1 mice were purchased from Inotiv and maintained at the University of Exeter.Mice were treated subcutaneously with 100 µl of Immunization Grade Chick Type II Collagen (Chondrex; 200 µg per mouse) in Complete Freund's Adjuvant (Sigma-Aldrich) at day 0 and with 100 µl of Immunization Grade Chick Type II Collagen (200 µg per mouse) in Incomplete Freund's Adjuvant (Sigma-Aldrich) at day 14, followed by an i.p. injection of 50 µg of LPS at day 26.Of anti-MICL antibodies, 0.7 mg of isotype control antibodies or PBS were administered i.p. every 48 h from day 17 to day 33.Mice were culled at day 35.In our animal facility, naive DBA/1 animals did not develop any spontaneous form of arthritis by 13 weeks of age (the latest timepoint in our experiments).

Histology
For histology, paws were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C and decalcified in 10% EDTA for 3-4 weeks.Decalcified paws were embedded in optimal cutting temperature (OCT) and cryosectioned or embedded in paraffin wax and sectioned before staining with haematoxylin and eosin, or Safranin O, haematoxylin and fast green.Scoring of histological sections was performed blinded using an arthritis severity score as previously described 18 .

Immunofluorescence staining protocol
Frozen tissue sections were thawed for 30 min at room temperature and washed with PBS before permeabilization with 0.25% Triton X-100 or 0.5% saponin in PBS for 10 min.After permeabilization, sections were washed with PBS again and blocked with 3% BSA at room temperature for 30 min.Tissue sections were stained with primary antibodies (anti-cit-H3 (Abcam), anti-DNA/H1 (Merck) and anti-GR-1 (produced in-house 59 )) diluted in blocking buffer (3% BSA in PBS) for 1 h 30 min in a humidified chamber, after which they were washed with PBS-Tween-20 (0.05%) and stained with secondary antibodies for 1 h at room temperature in the dark.Sections were washed as before and counterstained with 1 µg ml −1 DAPI and mounted in Vectashield antifade mounting medium for fluorescence (Vectorlabs).Coverslips were sealed with nail polish and slides were stored in the dark at 4 °C until imaging.Fluorescence was visualised using the Zeiss confocal LSM700 microscope and Zen Black software (Zeiss).The quantification of positive area (area + %) for each channel was conducted using Fiji software.Image preprocessing included utilizing the built-in 'Moments' algorithm for thresholding each channel.

Neutrophil isolation
Bone marrow neutrophils were isolated using Histopaque (Merck) by a density gradient centrifugation method or using the EasySep Mouse Neutrophil Enrichment Kit (StemCell Technologies) according to the manufacturer's guidelines.Flow cytometry analysis confirmed an approximately 90% pure neutrophil population (the remaining population consisting primarily of monocytes).To isolate thioglycollate-elicited neutrophils, mice were injected i.p. with 1 ml of 3% thioglycollate broth.After 4 h, mice were culled and neutrophils were harvested by peritoneal lavage with PBS 5 mM EDTA.
Human neutrophils from the blood of healthy donors were purified using a Ficoll-Paque density centrifugation method 60 or using the EasySep direct human neutrophil isolation kit (StemCell Technologies) as per the manufacturer's instructions.Samples were obtained from consenting healthy donors with the approval of the Faculty of Health and Life Sciences ethics review board, University of Exeter (eCLES-Bio000371) and the College of Life Sciences and Medicine ethics review board, University of Aberdeen (CERB number 1243).

NETs
Bone marrow and thioglycollate-elicited neutrophils were isolated as described above.Cells were resuspended in RPMI (without phenol red; Thermo Fisher Scientific) supplemented with 2% DNase −/− mouse serum and seeded in an eight-well iBidi µ-slide (iBidi).Cells were stimulated with 100 µg ml −1 of MSU crystals (Invivogen), 100 nM PMA (Sigma), isolated NETs (100 µg ml −1 based on NET-DNA concentration) or A. fumigatus hyphae and incubated at 37 °C with 5% CO 2 for 4 h.In some experiments, as indicated in the text, DPI (10 µM; Sigma) or NSC-87877 (5 µM; Cayman Chemical) were included in the assays.Extracellular DNA was visualized with 5 µM Sytox Green (Invitrogen).In experiments without fixation, cells were previously stained with Draq5 (Thermo Fisher Scientific) and cell-impermeable Sytox Green, and images were acquired on an inverted Zeiss AxioObserver Z1 using a PlanApo ×20/0.75NA dry lens (Carl Zeiss) and a Hamamatsu Fusion sCMOS camera with an attached incubation chamber (PeCon GmbH) at 37 °C.Fluorescent images were analysed in Image J, and NETs defined as Sytox Green-positive cells showing extrusions were counted.
NET (%) = (total Sytox Green-positive cells extruding NETs/total cells counted) × 100 For kinetic curves of NET formation, neutrophils were seeded in a 96-well black plate with a transparent bottom and the cells were left to adhere for 30 min in a cell culture incubator.Cells were stimulated with the defined reagents and 5 µM Sytox Green.Fluorescence signal (504-nm excitation and 523-nm emission) was measured every 10 min for 7 h in a Spark Cyto (Tecan) at 37 °C with 5% CO 2 .
NETs were isolated as previously described 61 .In brief, bone marrow-isolated or purified human neutrophils were plated in a six-well plate at a density of 1 × 10 6 cells per well in RPMI without phenol red.Following stimulation with 100 nM PMA for 4 h at 37 °C, the culture medium was removed and NETs were partially digested by application of a restriction enzyme mix combining the enzymes BseRI, PacI, NdeI and AfIII (New England Biolabs) at a concentration of 5 U ml −1 in NEB buffer for 1 h at 37 °C.Supernatants were collected and centrifuged at 300g for 10 min at 4 °C.NET supernatants were transferred to a fresh tube and stored at −80 °C until used.When indicated, NETs were treated with DNase I (Thermo Fisher Scientific) or proteinase K (Roche) for 1 h at 37 °C.
Recognition of isolated NETs by MICL was assessed by ELISA.A 96-well plate (Nunc Maxisorp) was coated with NETs diluted in PBS overnight at 4 °C.Wells were washed and blocked with blocking buffer (5% BSA) at room temperature.Fc-mMICL or Fc-mCLEC12B at 1 µg ml −1 in blocking buffer was added and incubated for 2 h at room temperature.Bound Fc-fusion proteins were detected with horseradish peroxidase-conjugated goat anti-human IgG ( Jackson Immunoresearch) diluted 1:10,000 for 1 h.TMB substrate was added, and absorbance was measured using a plate reader (Tecan).Purification of Fc-MICL and Fc-mCLEC12B, from transduced HEK293T cells (originally purchased from the American Type Culture Collection (ATCC), but not tested for Mycoplasma contamination for the experiments detailed in this paper), was performed as previously described 32 .
To analyse the recognition of isolated NETs by MICL using a MICLexpressing cell line, a 96-well plate was coated with NETs diluted in PBS overnight at 4 °C.Wells were washed, and BWZ.36 NFAT-LacZ cells (provided by W. Yokoyama; not tested for Mycoplasma contamination for the experiments detailed in this paper) expressing the CD3ζ chain fused to the transmembrane and carbohydrate recognition domain (CRD) of mMICL or mCLEC12B were added (2 × 10 5 cells per well) for 18 h at 37 °C.After stimulation, cells were centrifuged at 800g for 2 min and washed two times with PBS.Of CPRG substrate buffer, 100 µl was added per well.The reaction was stopped 4 h later with the addition of 50 µl of glycine-EDTA buffer, and the absorbance was measured using a plate reader 62 (Tecan).Antibody crosslinking with the appropriate receptor antibody was used to confirm the functionality of the chimeric receptor constructs.
To analyse neutrophil infiltration by isolated NETs, 6-8-week-old Micl −/− and C57BL/6J mice were injected i.p. with 300 µg NETs or 50 µg LPS.Four hours later, mice were euthanized, and their peritoneal cells were counted and analysed by flow cytometry.Cell-free DNA was evaluated using the Quant-iT PicoGreen dsDNA Assay (Invitrogen) following the manufacturer's instructions.

Immunofluorescent staining of in vitro samples
Neutrophils (5 × 10 4 ) were plated in an eight-well iBidi µ-slide (iBidi) 1 h before stimulation with isolated NETs, PMA or MSU.Four hours after stimulation, cells were fixed for 20 min at 37 °C with 2% PFA and permeabilized with 0.5% Triton X-100 in PBS.Samples were blocked with 3% (v/v) normal goat serum, 1% (w/v) BSA in PBS and incubated with anti-cit-H3 (Abcam) and anti-myeloperoxidase (R&D) antibodies.The secondary antibodies donkey anti-rabbit Cy5 and rabbit antigoat Alexa Fluor 488 were used.Finally, the samples were stained with DAPI.
Fluorescence quantification was conducted by segmenting the DAPI channel using QuPath software (version 0.4.3) with the Cellpose extension (https://github.com/BIOP/qupath-extension-cellpose).Before segmentation, preprocessing of the DAPI channel was carried out using Fiji software to mitigate background noise, involving background subtraction and enhanced contrast built-in functions.

Article
Preprocessing of the GFP and Texas Red channels involved a background subtraction of the mean grey value and two times the standard deviation of an empty region (background noise).The quantification of each channel was conducted using the measurement function of Fiji software, utilizing the mask generated by Qupath.

Imaging flow cytometry
Single cells isolated from the arthritic joint, as detailed above, were fixed in 1% PFA for 30 min at 37 °C.Cells were washed with PBS 2 mM EDTA for 5 min at 500g and stained with conjugated antibodies (detailed above), anti-DNA/histone 1 (Merck Millipore; 1.4 µg ml −1 ) and anti-cit-H3 (Abcam; diluted 1:300) for 1 h at room temperature.The secondary antibodies goat anti-rabbit APC (Molecular probes) and goat anti-mouse AF488 (Invitrogen) at 1 µg ml −1 were added for 1 h at 4 °C.Cells were washed and resuspended in 50 µl of PBS 2 mM EDTA before acquisition using the Amnis ImageStreamX MKII (Luminex) INSPIRE acquisition software.Files were analysed using IDEAS software v6.2 (Luminex).
Single cells were selected by plotting the area feature of brightfield channel 1 (BF1) versus the aspect ratio parameter of the same brightfield channel, which is the ratio of minor axis to the major axis of the applied mask, and describes the shape of the mask applied to the cells (Extended Data Fig. 7f).Focused cells were then selected by plotting the 'Gradient RMS' feature of BF1 against the BF1 contrast parameter.Cells with high-gradient and high-contrast value were more in focus and chosen for further analysis.All focused cells were used in the analysis for the presence of the cellular fluorescence parameters.Fluorescence parameters were measured in channel 2 (DNA/histone H1-AF488), channel 11 (cit-H3-APC) and channel 7 (Ly6G-BV421) with magnification set to ×40.

Serum collection from patients with rheumatoid arthritis
Serum samples from patients with rheumatoid arthritis were obtained from the SERA cohort 42 (Extended Data Table 1).Sera from healthy controls were obtained by collection of whole blood in a BD vacutainer serum collection tube (BD Biosciences).The blood was allowed to clot for 15-30 min at room temperature, after which the tubes were centrifuged at 2,000g for 10 min in a 4 °C.Sera were removed, aliquoted and stored at −80 °C.Serum samples were obtained from consenting healthy donors with the approval of the College of Life Sciences and Medicine ethics review board, University of Aberdeen (CERB number 1243).

Serum collection from patients with COVID-19
Blood from 121 patients diagnosed with SARS-CoV-2 infection at Weill-Cornell Medicine between March and July 2020.Research on patients with COVID-19 was reviewed and approved by the Institutional Review Board of Weill-Cornell Medicine (New York Presbyterian and Lower Manhattan hospitals; #IRB 20-03021645 and #IRB 20-03021671).Informed consents were obtained from all enrolled patients and health-care workers by trained staff, and records were maintained in our research database for the duration of the study.All patients were classified as mild/moderate (n = 25) and severe (n = 66) disease according to oxygen requirements with mild/moderate disease defined as SARS-CoV-2 infection and less than 6 l of non-invasive supplementary oxygen to maintain SpO2 > 92%, and severe disease was defined as SARS-CoV-2 infection requiring hospitalization and received 6 l or more supplementary oxygen or mechanical ventilation.For controls, we used blood samples from 36 SARS-CoV-2-negative individuals collected by the JRI IBD Live Cell Bank Consortium at Weill-Cornell Medicine.Heparinized plasma and serum samples were aliquoted, heat-inactivated at 56 °C for 1 h and then stored at −80 °C.

Serum collection from patients with SLE
All patients with SLE in the study met the revised American College of Rheumatology criteria 64 and the SLICC criteria 65 .Some patients had a history of biopsy-proven nephritis according to the International Society of Nephrology/Renal Pathology Society classification.Healthy female volunteers (with no family history of autoimmune disease) served as age-matched and ethnicity-matched controls.All patients provided informed consent, and samples used in this research project were obtained from the Imperial College Healthcare Tissue Bank (ICHTB).The ICHTB is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London.The ICHTB is approved by Wales REC3 to release human material for research (22/WA/0214), and the samples for this project (ref: R13010a) were issued from sub-collection reference number IMM_MB_13_001.

ELISAs from human patients
Sera from patients with rheumatoid arthritis, COVID-19 and SLE and healthy controls were screened for the presence of MICL autoantibodies using a modified ELISA method 25 .In brief, Nunc Maxisorp 96-well plates were coated with equivalent amounts of the Fc-fusion proteins Fc-hMICL and Fc-mCLEC12B diluted in PBS overnight at 4 °C.Plates were blocked with 10% BSA and 10% HI goat serum (Merck) in PBS for 1 h.Sera from patients with rheumatoid arthritis, COVID-19 and SLE and healthy control were diluted to a 1:256 dilution in PBS and added to the pre-blocked plate and incubated for 2 h.Bound autoantibodies were detected with horseradish peroxidase-conjugated goat anti-human F(ab′)2 fragment ( Jackson Immunoresearch) diluted 1:50,000 in PBS for 1 h.TMB substrate was added, and absorbance was measured at 450 nm in a plate reader (Tecan).

A. fumigatus systemic infection model
Micl −/− and C57BL/6J female mice were injected intravenously with 10 6 A. fumigatus ATCC 13073 conidia, as previously described 66 .Mice were culled when they lost 20% body weight or had become moribund.To investigate the role of NET formation, mice were injected i.p. with 4 mg kg −1 daily of GSK484 (Cambridge Biosciences) from day 1 to day 5. Organs were homogenized in PBS and used for the determination of fungal burdens and levels of inflammatory cytokines.Fungal burdens were determined by serial dilution onto potato dextrose agar plates and normalized to organ weights.Cytokines were measured by ELISA (BD Biosciences), as described by the manufacturer, and normalized to protein concentration.

Statistical analysis
Data are represented as mean ± s.d., unless otherwise indicated.All statistical analyses were performed using GraphPad Prism (v9, Graph-Pad Software) and depicted in the respective figure legends.For all experiments with two groups, two-tailed unpaired Student's t-tests (equal variances) or two-tailed Mann-Whitney tests were used.One-way or two-way ANOVA (with equal variances) with correction for multiple comparisons was performed for experiments with more than two groups.All P < 0.05 were considered statistically significant.

Reporting summary
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Life sciences study design
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Sample size
Sample sizes of at least five per group were chosen as this would allow the detection of a 25% difference in the mean between experimental and control groups with a probability of greater than 95% (p < 0.05), assuming a standard deviation of around 15% and a minimum power value of 0.8.Randomization Mouse experiments were performed by random assignation of age-and sex-matched mice in experimental groups at the beginning of each experiment to experimental or control groups, females were co-housed, and experiments were not blinded.For in vitro experiments, samples were randomly assigned to experimental or control groups.

Blinding
Experiments were not blinded, as the investigator who planned the experiments, also performed them but were conducted according to standardized protocols and procedures.
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Fig. 1 |
Fig. 1 | MICL is required for control of neutrophil responses.a, tSNE plots of CD45 + myeloid populations in the inflamed ankle joint during CAIA displayed as CD11b + cells (grey), neutrophils (blue), Ly6C high cells (green), Ly6C low cells (orange) and remaining antigen-presenting cells (APCs; pale violet).b, Myeloid cell populations (defined as shown in the gating strategy in Extended Data Fig. 1b) in the inflamed ankle joint during CAIA at day 7 are represented as a percentage of total live cells (pooled data from two independent experiments with four mice per group per experiment).WT versus Micl −/− neutrophils P < 0.0001.c, Schematic representation of the anti-Ly6G-mediated neutrophil depletion strategy in the CAIA model.D0, day 0; LPS, lipopolysaccharide.d, Quantification of neutrophils (CD45 + CD11b + F4/80 − SSC high ) in the peripheral blood at day 9 by flow cytometry (n = 1 experiment with 3 mice per group).Isotype versus anti-Ly6G WT P = 0.0035 and Micl −/− P = 0.0021.e, Severity scoring of WT and knockout (Micl −/− ) mice treated with isotype or anti-Ly6G antibodies (Abs), as indicated (n = 1 experiment with 5 mice per group).f, Schematic representation of neutrophil adoptive transfer during CAIA in WT mice.KO, knockout.g, CAIA severity scoring in WT mice that received adoptively transferred WT or knockout neutrophils, as indicated (pooled data from two independent experiments with five mice per group per experiment).Day 8 WT versus Micl −/− P = 0.0013.h, Representative Safranin O-stained sections of the tarsal joints of WT mice that received adoptively transferred WT or knockout neutrophils, as indicated at day 8 (left).Synovial inflammation (black asterisks) is indicated.Scale bars, 500 µm.The histological arthritis severity score is also shown (right; nine mice per group).WT versus Micl −/− P = 0.0281.Data are represented as mean ± s.d.(b,d,e,g,h).Statistical significance was determined by two-way analysis of variance (ANOVA) with Bonferroni's multiple comparisons test (b,d,e,g).Data were analysed using an unpaired two-tailed Student's t-test (h).*P < 0.05, **P < 0.01 and ****P < 0.0001.Schematics in panels c,f were created using BioRender (https://biorender.com).

Fig. 3 |
Fig. 3 | Anti-MICL antibodies modulate neutrophil function and correlate with disease.a, ROS generation by MSU or PMA of human neutrophils (hPMNs) in the presence or absence of anti-MICL antibodies (anti-hMICL), depicted as RLU over time.Anti-hMICL versus isotype MSU P = 0.0084.b, Fluorescence of NET-bound Sytox green of MSU or PMA-induced NETs in hPMNS in the presence or absence of anti-hMICL.MSU anti-hMICL versus isotype P = 0.0043.RFU, relative fluorescence units.c, ROS generation by MSU of hPMNs in the presence of serum from patients with rheumatoid arthritis (RA) and healthy controls (HC).HC1 versus RA38 P < 0.0001, HC1 versus RA40 P < 0.0001, HC2 versus RA38 P < 0.0001 and HC2 versus RA40 P < 0.0001.d, Level of anti-MICL autoantibodies detected in serum samples from patients with rheumatoid arthritis (n = 199) and healthy controls (n = 132).Abs 450 , absorbance at 450 nm.e, Correlation of MICL autoantibody titres with rheumatoid factor levels in SERA cohort serum from patients with rheumatoid arthritis.f, Correlation of MICL autoantibody titres with CCP levels in SERA cohort serum from patients with rheumatoid arthritis.g, Level of anti-MICL autoantibodies detected in serum from patients with SLE (n = 40) and healthy controls (n = 25).h, Level of anti-MICL autoantibodies detected in serum from patients with mild/moderate (mCOVID-19; n = 25) and severe (sCOVID-19; n = 67) COVID-19 and healthy controls (n = 36).sCOVID-19 versus mCOVID-19 P = 0.0472 and sCOVID-19 versus healthy controls P = 0.0284.i, ROS generation and area under curve (AUC) of hPMNs stimulated with MSU in the presence of serum from patients with SLE pre-incubated with Fc-hMICL.Data are a representative example of n = 3 (a,b) or n = 2 (c,i) independent experiments, and mean ± s.d.performed in triplicate.The AUC was analysed by one-way ANOVA with Tukey's multiple comparisons test.Box plots extend from the 25th to 75th percentile, including the median, and whiskers extend from the minimum to maximum value (d,g,h).Data were analysed by unpaired two-tailed Student's t-test (d,g), a Spearman correlation and two-sided t-test (e,f), or using one-way ANOVA with Kruskal-Wallis multiple comparisons test (h).*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.

Fig. 4 |
Fig. 4 | NET-MICL interaction regulates inflammation.a, ROS generation by bone marrow neutrophils stimulated with preformed NETs, depicted as RLU over time.Data are a representative example of n = 4 independent experiments, depicted as mean ± s.d.performed in triplicate.mNET, preformed mouse NETs.b, Immunofluorescence staining for MPO (yellow), cit-H3 (magenta) and DNA (DAPI; grey) of neutrophils stimulated with preformed NETs.Scale bars, 200 µm.Quantification is in Extended Data Fig. 7b.c, ROS generation by human neutrophils stimulated with preformed NETs in the presence or absence of antibodies targeting MICL.Data are a representative example of n = 2 independent experiments, depicted as mean ± s.d.performed in duplicate.hPMNs versus anti-hMICL P = 0.0011

Fig. 5 |
Fig. 5 | MICL regulates NET formation during fungal infection.a, Fluorescence of A. fumigatus hyphae-induced NETs bound to Sytox green in bone marrowisolated neutrophils from WT and MICL-deficient mice or human neutrophils in the presence of antibodies to MICL.The fluorescence background signal from the unstimulated controls was subtracted from values.Data are a representative example of n = 3 independent experiments, depicted as mean ± s.d.performed in triplicate.The AUC was analysed using an unpaired two-tailed Student's t-test.WT versus Micl −/− P = 0.0272 and anti-hMICL versus isotype P = 0.0032.b, Survival of mice following intravenous infection with 10 6 A. fumigatus conidia (n = 15 mice per group).Pooled data are from two independent experiments, analysed by log-rank test; P = 0.0005.c-e, Brain fungal burdens (c), and serum (d) and brain (e) cytokine levels of mice 2 days after intravenous infection with 10 6 A. fumigatus conidia.Values are mean ± s.e.m. of pooled data from two independent

Extended Data Fig. 2 |
MICL is required for controling neutrophil responses during arthritis.a, Schematic of K/BxN serum transfer model and severity scoring shown as mean ± SD analysed over time.b, Total live cell populations (defined by gating strategy in Extended Data Fig.1b) isolated from inflamed joints on day 10, mean ± SD.Neutrophils WT vs MICL −/− p = 0.0086.a,b, Data is a representative example of three independent experiments, with 4 mice/group/ experiment, and analysed using two-way ANOVA with Bonferroni's multiple comparisons test.Schematic in panel a was created using BioRender (https:// biorender.com).c,d, tSNE plots of CD45 + myeloid populations displayed as neutrophils (blue), Ly6C high cells (green), Ly6C low cells (orange), antigen presenting cells (APCs) (pale violet), and all other subpopulation (grey) in the bone marrow (c), and blood (d) of naïve mice.Data is a representative example of three independent experiments, with 4 mice/group/experiment, represented as mean ± SD. e,f, Fold change in mean fluorescent intensity (MFI), relative to wild-type mice of (e) neutrophil activation markers (CD11b, CD18, CD62L) and (f) chemokine receptors (CXCR2, CCR1, C5aR), isolated concomitantly from joints or blood of arthritic mice during CAIA on day 7 (pooled data from two independent, n = 10 biologically independent mice).g, Fold change in mean fluorescence intensity (MFI) on neutrophils, relative to wild-type mice of CD11b, CD18, CD62L and CCR1, isolated from the joints of arthritic mice on day 10.Data is a representative example of n = 3 independent experiments with 4 mice/group/experiment, shown as mean ± SD and analysed using two-way ANOVA with Bonferroni's multiple comparisons test.h,j, Fold change in mean fluorescent intensity (MFI) relative to wild-type mice of neutrophil activation markers (CD11b, CD18, CD62L) and chemokine receptors (CXCR2, CCR1), isolated concomitantly from bone marrow (h) and blood ( j) of naïve mice (representative example of n = 2 experiments with 5 mice/group/experiment).WT, wild-type mice; KO, MICL −/− mice; *p < 0.05; ns, not significant.Extended DataFig. 4 | MICL regulates NET formation.a, Fluorescence of NET-bound Sytox green of MSU or PMA-induced NETs in thioglycollate-elicited neutrophils analysed using a SPARK CYTO reader (Tecan) every 10 min for up to 7 h.Data is a representative example of n = 3 independent experiments, mean ± SD performed in triplicate.b, Representative Sytox green fluorescent (cyan), Draq5 (magenta), and bright field (grey) images of MSU-induced NETs in bone marrow neutrophils isolated from WT and MICL-deficient animals.Cells were stained unfixed.Quantification (as percentage of Sytox green positive cells extruding NETs) shown as mean ± SD, n = 1 experiment performed in triplicate.Statistical significance determined by Student's t-test.c, Representative Sytox green fluorescent and bright field images of MSU-induced NETs in thioglycollateelicited neutrophils isolated from MICL-deficient animals with and without DPI pre-treatment.Scale bar = 100 µm.Quantification (mean ± SD) shown right (n = 2 experiments performed in triplicate).Statistical significance determined by Student's t-test.d, Fluorescence of NET-bound Sytox of MSU-induced NETs in thioglycollate-elicited neutrophils with and without NSC87877 treatment analysed 4 h after stimulation.Pooled data from two independent experiments performed in duplicate represented as mean ± SD. e, Representative confocal immunofluorescence microscopy images of NETs in CAIA WT and MICL −/− synovial sections on day 11 (as per schedule in Extended Data Fig. 1a).GR-1 (purple), DAPI (blue), Cit-H3 (yellow), and DNA/H1 (green) and quantification of cit-H3/GR-1 area (n = 1 experiment with 3 mice/group).NETs are defined as GR-1+Cit-H3 + DNA/H1+ stained cells.Scale bar = 100 µm.Statistical significance determined by Student's t-test.f, Representative imaging flow cytometry examples of NET positive (+) and NET negative (−) neutrophils isolated from the ankle joints of mice at day 11 during CAIA.BF, Brightfield; DNA/H1 (green); Ly6G (purple); Cit-H3 (red).*, p < 0.05.Extended Data Fig. 7 | MICL recognizes NETs.a, NET-bound Sytox green fluorescence of bone marrow-isolated neutrophils stimulated with preformed murine NETs (mNETs).Data is a representative example of n = 3 independent experiments, mean ± SD performed in triplicate.b, Mean fluorescence intensity (MFI) of cit-H3 and MPO (mean ± SD, 3 fields of view per condition) during NET formation in WT or MICL-deficient mNETs-stimulated neutrophils.Statistical significance determined by Student's t-test.c, ROS production of MICL-deficient neutrophils mNETs-stimulated with or without polyxymin B. Data is a representative example of n = 2 independent experiments, mean ± SD performed in triplicate.d, NET formation and quantification of area under curve (AUC, right) of WT or MICL-deficient mNETs-stimulated neutrophils with GSK484 or DPI.Data is a representative example of n = 2 independent experiments, mean ± SD performed in triplicate.e, Fold change in MFI of MICL expression on cultured neutrophils (CD66b + CD15 + ), relative to human neutrophils treated with negative control scrambled guide RNA.f, ROS production by preformed NETs of human MICL-knockout cultured neutrophils (hKO) or human WT derived from CD34 + haematopoietic progenitors.Data is a representative example of n = 2 independent experiments, mean ± SD performed in triplicate.g, Fc-MICL NET recognition by ELISA in the presence/ absence of anti-MICL antibodies.Pooled data represented as mean ± SD of n = 2 independent experiments performed in triplicate.Statistical significance determined by One-way ANOVA and Bonferroni multiple comparison test.h, ROS production of MICL-deficient neutrophils stimulated with untreated NETs, Proteinase K-treated NETs or DNaseI-treated NETs.Data is a representative example of n = 3 independent experiments, mean ± SD performed in triplicate.i, Fc-MICL genomic DNA recognition by ELISA in the presence/absence of anti-MICL antibodies.Pooled data represented as mean ± SD of n = 2 independent experiments performed in triplicate.Statistical significance determined by One-way ANOVA and Bonferroni multiple comparison test.j, Cell free DNA concentration 4 h after NETs or LPS intraperioteneal injection in WT and MICL-deficient animals (n = 1 experiment with 6 animals/group).*, p < 0.05.Extended Data Fig. 8 | MICL regulates NET formation during fungal infection.a, Representative Sytox green fluorescent (DNA, green) and calcofluor white (blue) images of A. fumigatus-induced NETs in bone marrow neutrophils isolated from WT or MICL-deficient animals and quantification as percentage of neutrophils under NETosis (3 fields of view per condition) at 4 h post-stimulation.Statistical significance determined by student's t-test.b, Tissue fungal burdens of mice two days after intravenous (i.v) infection with 10 6 A. fumigatus conidia (values shown are mean± SEM of pooled data from two independent experiments, n = 14 biologically independent mice).c, Survival of mice following i.v infection with 10 6 A. fumigatus conidia and treated with vehicle control (n = 1 experiment with 6 mice/group) analysed by log-rank test.p = 0.0195.CFU, colony-forming unit.*, p < 0.05.Extended Data Table 1 | Patient characteristics of the SERA cohort nature portfolio | reporting summary April 2023 G, Zhao Z, Rand S, Doron I, Putzel GG, Worgall S, Cushing M, Westblade L, Inghirami G, Parkhurst CN, Guo CJ, Schotsaert M, García-Sastre A, Josefowicz SZ, Salvatore M, Iliev ID.Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.Nat Immunol.2023 Nov;24(11):1879-1889. doi: 10.1038/s41590-023-01637-4)For the SLE cohort, all patients gave informed consent and samples used in this research project were obtained from the Imperial College Healthcare Tissue Bank (ICHTB).ICHTB is supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London.ICHTB is approved by Wales REC3 to release human material for research (22/WA/0214), and the samples for this project (Ref: R13010a) were issued from sub-collection reference number IMM_MB_13_001.
Fig 1a.One mouse was excluded from analysis due to lack of response Extended data Fig 6c. 3 animals were excluded since they were euthanized during the experiment Replication All experiments were independently replicated at least once unless otherwise indicated in the manuscript.

,
Fluorescence of A. fumigatus hyphae-induced NETs bound to Sytox green in bone marrowisolated neutrophils from WT and MICL-deficient mice or human neutrophils in the presence of antibodies to MICL.The fluorescence background signal from the unstimulated controls was subtracted from values.Data are a representative example of n = 3 independent experiments, depicted as mean ± s.d.performed in triplicate.The AUC was analysed using an unpaired two-tailed Student's t-test.(d,e) biologically independent mice, analysed using an unpaired two-tailed Student's t-test.IL-6 WT versus Micl −/− P = 0.0185 (d) and G-CSF WT versus Micl −/− P = 0.0492 (e).CFU, colony-forming unit.f, Survival of mice following intravenous infection with 10 6 A. fumigatus conidia and treated with GSK484 (PAD4 inhibitor).Pooled data are from two independent experiments; n = 14 biologically independent mice, analysed by log-rank test.g, Brain fungal burdens of mice 2 days after intravenous infection with 10 6 A. fumigatus conidia treated with GSK484 (PAD4 inhibitor) or vehicle control.Values are mean ± s.e.m. of 1 experiment with n = 6 biologically independent mice, analysed by one-way ANOVA with Tukey's multiple comparisons test.