Alpha1-antitrypsin impacts innate host-pathogen interactions with Candida albicans by stimulating fungal filamentation.

Our immune system possesses sophisticated mechanisms to cope with invading microorganisms, while pathogens evolve strategies to deal with threats imposed by host immunity. Human plasma protein α1-antitrypsin (AAT) exhibits pleiotropic immune-modulating properties by both preventing immunopathology and improving antimicrobial host defence. Genetic associations suggested a role for AAT in candidemia, the most frequent fungal blood stream infection in intensive care units, yet little is known about how AAT influences interactions between Candida albicans and the immune system. Here, we show that AAT differentially impacts fungal killing by innate phagocytes. We observed that AAT induces fungal transcriptional reprogramming, associated with cell wall remodelling and downregulation of filamentation repressors. At low concentrations, the cell-wall remodelling induced by AAT increased immunogenic ß-glucan exposure and consequently improved fungal clearance by monocytes. Contrastingly, higher AAT concentrations led to excessive C. albicans filamentation and thus promoted fungal immune escape from monocytes and macrophages. This underscores that fungal adaptations to the host protein AAT can differentially define the outcome of encounters with innate immune cells, either contributing to improved immune recognition or fungal immune escape.

S100A8/A9 Is a Marker for the Release of Neutrophil Extracellular Traps and Induces Neutrophil Activation.

Neutrophils are the most abundant innate immune cells in the circulation and they are the first cells recruited to sites of infection or inflammation. Almost half of the intracellular protein content in neutrophils consists of S100A8 and S100A9, though there has been controversy about their actual localization. Once released extracellularly, these proteins are thought to act as damage-associated molecular patterns (DAMPs), though their mechanism of action is not well understood. These S100 proteins mainly form heterodimers (S100A8/A9, also known as calprotectin) and this heterocomplex is recognized as a useful biomarker for several inflammatory diseases. We observed that S100A8/A9 is highly present in the cytoplasmic fraction of neutrophils and is not part of the granule content. Furthermore, we found that S100A8/A9 was not released in parallel with granular content but upon the formation of neutrophil extracellular traps (NETs). Accordingly, neutrophils of patients with chronic granulomatous disease, who are deficient in phorbol 12-myristate 13-acetate (PMA)-induced NETosis, did not release S100A8/A9 upon PMA stimulation. Moreover, we purified S100A8/A9 from the cytoplasmic fraction of neutrophils and found that S100A8/A9 could induce neutrophil activation, including adhesion and CD11b upregulation, indicating that this DAMP might amplify neutrophil activation.

Formation of neutrophil extracellular traps requires actin cytoskeleton rearrangements.

Neutrophils are important effector cells in the host defense against invading microorganisms. One of the mechanisms they use to eliminate pathogens is the release of neutrophil extracellular traps (NETs). Although NET release and subsequent cell death known as NETosis have been intensively studied, the cellular components and factors determining or facilitating the formation of NETs remain incompletely understood. Using various actin polymerization and myosin II modulators on neutrophils from healthy individuals, we show that intact F-actin dynamics and myosin II function are essential for NET formation when induced by different stimuli; that is, phorbol 12-myristate 13-acetate, monosodium urate crystals, and Candida albicans. The role of actin polymerization in NET formation could not be explained by the lack of reactive oxygen species production or granule release, which were normal or enhanced under the given conditions. Neutrophils from patients with very rare inherited actin polymerization defects by either actin-related protein 2/3 complex subunit 1B or megakaryoblastic leukemia 1 deficiency also failed to show NETosis. We found that upon inhibition of actin dynamics, there is a lack of translocation of neutrophil elastase to the nucleus, which may explain the impaired NET formation. Collectively, our data show the essential requirement of an intact and active actin polymerization process, as well as active myosin II to enable the release of nuclear DNA by neutrophils during NET formation.

Molecular Mechanisms of Leukocyte Migration and Its Potential Targeting-Lessons Learned From MKL1/SRF-Related Primary Immunodeficiency Diseases.

Megakaryoblastic leukemia 1 (MKL1) deficiency is one of the most recently discovered primary immunodeficiencies (PIDs) caused by cytoskeletal abnormalities. These immunological "actinopathies" primarily affect hematopoietic cells, resulting in defects in both the innate immune system (phagocyte defects) and adaptive immune system (T-cell and B-cell defects). MKL1 is a transcriptional coactivator that operates together with serum response factor (SRF) to regulate gene transcription. The MKL/SRF pathway has been originally described to have important functions in actin regulation in cells. Recent results indicate that MKL1 also has very important roles in immune cells, and that MKL1 deficiency results in an immunodeficiency affecting the migration and function of primarily myeloid cells such as neutrophils. Interestingly, several actinopathies are caused by mutations in genes which are recognized MKL(1/2)-dependent SRF-target genes, namely ACTB, WIPF1, WDR1, and MSN. Here we summarize these and related (ARPC1B) actinopathies and their effects on immune cell function, especially focusing on their effects on leukocyte adhesion and migration. Furthermore, we summarize recent therapeutic efforts targeting the MKL/SRF pathway in disease.

Neutrophil specific granule and NETosis defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.

When Actin is Not Actin' Like It Should: A New Category of Distinct Primary Immunodeficiency Disorders.

An increasing number of primary immunodeficiencies (PIDs) have been identified over the last decade, which are caused by deleterious mutations in genes encoding for proteins involved in actin cytoskeleton regulation. These mutations primarily affect hematopoietic cells and lead to defective function of immune cells, such as impaired motility, signaling, proliferative capacity, and defective antimicrobial host defense. Here, we review several of these immunological "actinopathies" and cover both clinical aspects, as well as cellular mechanisms of these PIDs. We focus in particular on the effect of these mutations on human neutrophil function.

Defective neutrophil development and specific granule deficiency caused by a homozygous splice-site mutation in SMARCD2.

BACKGROUND: SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) has recently been shown to have a critical role in granulopoiesis in humans, mice, and zebrafish. Our patient presented with delayed cord separation, failure to thrive, and sepsis. Retrospective whole-exome sequencing confirmed a homozygous splice-site mutation in SMARCD2. OBJECTIVE: We sought to provide the second description of human SMARCD2 deficiency and the first functional analysis of human primary SMARCD2-deficient cells. METHODS: Heparinized venous blood and bone marrow were collected from the patient after obtaining informed consent. Patient leukocytes and CD34+ cells were then isolated, phenotyped, and assessed functionally. RESULTS: Circulating neutrophils appeared phenotypically immature, lacking multilobed nuclei, and neutrophil granules lacked lactoferrin but showed normal levels of myeloperoxidase. Neutrophil oxidative burst was preserved in response to phorbol 12-myristate 13-acetate. Patient bone marrow-derived neutrophils and white blood cells showed a severely impaired chemotactic response. Furthermore, white blood cells showed defective in vitro killing of Staphylococcus aureus, consistent with a specific granule deficiency. Finally, patient bone marrow-derived CD34+ cells showed markedly impaired in vitro expansion and differentiation toward the neutrophil lineage. Before her molecular diagnosis, our patient underwent hematopoietic stem cell transplantation and is well 8 years later. CONCLUSIONS: This report highlights an important role for SMARCD2 in human myelopoiesis and the curative effect of hematopoietic stem cell transplantation for the hematopoietic features of SMARCD2 deficiency.

MKL1 deficiency results in a severe neutrophil motility defect due to impaired actin polymerization.

Megakaryoblastic leukemia 1 (MKL1) promotes the regulation of essential cell processes, including actin cytoskeletal dynamics, by coactivating serum response factor. Recently, the first human with MKL1 deficiency, leading to a novel primary immunodeficiency, was identified. We report a second family with 2 siblings with a homozygous frameshift mutation in MKL1. The index case died as an infant from progressive and severe pneumonia caused by Pseudomonas aeruginosa and poor wound healing. The younger sibling was preemptively transplanted shortly after birth. The immunodeficiency was marked by a pronounced actin polymerization defect and a strongly reduced motility and chemotactic response by MKL1-deficient neutrophils. In addition to the lack of MKL1, subsequent proteomic and transcriptomic analyses of patient neutrophils revealed actin and several actin-related proteins to be downregulated, confirming a role for MKL1 as a transcriptional coregulator. Degranulation was enhanced upon suboptimal neutrophil activation, whereas production of reactive oxygen species was normal. Neutrophil adhesion was intact but without proper spreading. The latter could explain the observed failure in firm adherence and transendothelial migration under flow conditions. No apparent defect in phagocytosis or bacterial killing was found. Also, monocyte-derived macrophages showed intact phagocytosis, and lymphocyte counts and proliferative capacity were normal. Nonhematopoietic primary fibroblasts demonstrated defective differentiation into myofibroblasts but normal migration and F-actin content, most likely as a result of compensatory mechanisms of MKL2, which is not expressed in neutrophils. Our findings extend current insight into the severe immune dysfunction in MKL1 deficiency, with cytoskeletal dysfunction and defective extravasation of neutrophils as the most prominent features.

Adjunctive interferon-γ immunotherapy in a pediatric case of Aspergillus terreus infection.

Aspergillus terreus causes invasive aspergillosis (IA) in immunocompromised patients. Treatment is complicated by intrinsic resistance to amphotericin B and thereby contributing to a high mortality. Therefore, we conducted in vitro studies to investigate the effectivity of adjunctive recombinant interferon-γ immunotherapy. We describe a pediatric patient with A. terreus IA who received adjunctive recombinant interferon-γ (rIFNγ) immunotherapy. In vitro studies were conducted to investigate the capacity of rIFNγ to improve antifungal host defense in terms of fungal killing ability and the release of pro-inflammatory cytokines in cells of the patient as well as healthy controls. An 8-year-old female pediatric patient with leukemia developed A. terreus IA. She clinically deteriorated and had high serum galactomannan levels despite broad antifungal therapy. Therefore, adjunctive immune stimulatory therapy with rIFNγ was initiated. After 3 weeks of treatment, galactomannan levels decreased and the patient clinically showed improvement. Addition of rIFNγ boosted the capacity of monocytes of healthy volunteers to mount TNFα and IL-1ß cytokine responses to Escherichia coli LPS, and increased TNFα response to both A. terreus and Aspergillus fumigatus. Monocytes isolated from the patient's blood demonstrated a similar augmented cytokine induction in response to rIFNγ. In addition, rIFNγ increased the capacity of monocytes from healthy volunteers as well as monocytes from the patient to kill A. terreus spores. Adjuvant immunotherapy with rIFNγ might be a promising additional treatment strategy that could be used to improve outcome in patients with refractory invasive A. terreus infections or other resistant invasive Aspergillus infections.

Toll-like receptor 2 induced cytotoxic T-lymphocyte-associated protein 4 regulates Aspergillus-induced regulatory T-cells with pro-inflammatory characteristics.

Patients with cystic fibrosis, chronic obstructive pulmonary disease, severe asthma, pre-existing pulmonary lesions, and severely immunocompromised patients are susceptible to develop infections with the opportunistic pathogenic fungus Aspergillus fumigatus, called aspergillosis. Infections in these patients are associated with persistent pro-inflammatory T-helper (TH)2 and TH17 responses. Regulatory T-cells, natural suppressor cells of the immune system, control pro-inflammatory T-cell responses, but can also contribute to disease by shifting to a pro-inflammatory TH17-like phenotype. Such a shift could play an important role in the detrimental immunopathology that is seen in aspergillosis. Our study demonstrates that Aspergillus fumigatus induces regulatory T-cells with a TH17-like phenotype. We also demonstrate that these regulatory T-cells with a pro-inflammatory TH17-like phenotype can be reprogrammed to their "classical" anti-inflammatory phenotype by activating Toll-like receptor 2 (TLR2), which regulates the induction of cytotoxic T-lymphocyte-associated protein 4 (CTLA4). Similarly, soluble CTLA4 could reverse the pro-inflammatory phenotype of Aspergillus-induced regulatory T-cells. In conclusion, our results suggest a role for regulatory T-cells with a pro-inflammatory TH17-like phenotype in Aspergillus-associated immunopathology, and identifies key players, i.e. TLR2 and CTLA4, involved in this mechanism.