ASSESSMENT OF CONGENITAL NEUTROPENIA IN CHILDREN: COMMON CLINICAL SCENERIES AND CLUES FOR MANAGEMENT

A disparate group of rare hematological diseases characterized by impaired maturation of neutrophil granulocytes defines congenital neutropenias. Neutropenic patients are prone to recurrent infections beginning in the first months of life. Of interest is “cyclic neutropenia”, an ultra-rare disorder revealed by sinusoidal variations of the neutrophil count and periodically-recurring infections every 21 days. Diagnosis of these disorders is frequently obscured by the multiple causes of recurrent fevers in children. Aim of this overview is to outline the physical assessment of children presenting with early-onset symptomatic neutropenia, identify the disease between the many medical conditions and even emergencies which should enter in differential diagnosis, hint at the potential management with granulocyte-colony stimulating factor, define the risk of evolution to hematologic malignancy, and summarize inter-professional team strategies for improving care coordination and outcomes of such patients.

SARS-CoV-2 and the Immune Response in Pregnancy with Delta Variant Considerations

As of September 2021, there has been a total of 123,633 confirmed cases of pregnant women with SARS-CoV-2 infection in the US according to the CDC, with maternal death being 2.85 times more likely, pre-eclampsia 1.33 times more likely, preterm birth 1.47 times more likely, still birth 2.84 times more likely, and NICU admission 4.89 times more likely when compared to pregnant women without COVID-19 infection. In our literature review, we have identified eight key changes in the immunological functioning of the pregnant body that may predispose the pregnant patient to both a greater susceptibility to SARS-CoV-2, as well as a more severe disease course. Factors that may impede immune clearance of SARS-CoV-2 include decreased levels of natural killer (NK) cells, Th1 CD4+ T cells, plasmacytoid dendritic cells (pDC), a decreased phagocytic index of neutrophil granulocytes and monocytes, as well as the immunomodulatory properties of progesterone, which is elevated in pregnancy. Factors that may exacerbate SARS-CoV-2 morbidity through hyperinflammatory states include increases in the complement system, which are linked to greater lung injury, as well as increases in TLR-1 and TLR-7, which are known to bind to the virus, leading to increased proinflammatory cytokines such as IL-6 and TNF-α, which are already elevated in normal pregnant physiology. Other considerations include an increase in angiotensin converting enzyme 2 (ACE2) in the maternal circulation, leading to increased viral binding on the host cell, as well as increased IL-6 and decreased regulatory T cells in pre-eclampsia. We also focus on how the Delta variant has had a concerning impact on SARS-CoV-2 cases in pregnancy, with an increased case volume and proportion of ICU admissions among the infected expecting mothers. We propose that the effects of the Delta variant are due to a combination of (1) the Delta variant itself being more transmissible, contagious, and efficient at infecting host cells, (2) initial evidence pointing to the Delta variant causing a significantly greater viral load that accumulates more rapidly in the respiratory system, (3) the pregnancy state being more susceptible to SARS-CoV-2 infection, as discussed in-depth, and (4) the lower rates of vaccination in pregnant women compared to the general population. In the face of continually evolving strains and the relatively low awareness of COVID-19 vaccination for pregnant women, it is imperative that we continue to push for global vaccine equity.

α1-Antitrypsin attenuates acute rejection of orthotopic murine lung allografts

Background α1-Antitrypsin (AAT) is an acute phase glycoprotein, a multifunctional protein with proteinase inhibitory, anti-inflammatory and cytoprotective properties. Both preclinical and clinical experiences show that the therapy with plasma purified AAT is beneficial for a broad spectrum of inflammatory conditions. The potential effects of AAT therapy have recently been highlighted in lung transplantation (LuTx) as well. Methods We used a murine fully mismatched orthotopic single LuTx model (BALB/CJ as donors and C57BL/6 as recipients). Human AAT preparations (5 mg, n = 10) or vehicle (n = 5) were injected to the recipients subcutaneously prior to and intraperitoneally immediately after the LuTx. No immune suppressive drugs were administered. Three days after the transplantation, the mice were sacrificed, and biological samples were assessed. Results Histological analysis revealed significantly more severe acute rejection in the transplanted lungs of controls than in AAT treated mice (p < 0.05). The proportion of neutrophil granulocytes, B cells and the total T helper cell populations did not differ between two groups. There was no significant difference in serum CXCL1 (KC) levels. However, when compared to controls, human AAT was detectable in the serum of mice treated with AAT and these mice had a higher serum anti-elastase activity, and significantly lower proportion of Th1 and Th17 among all Th cells. Cleaved caspase-3-positive cells were scarce but significantly less abundant in allografts from recipients treated with AAT as compared to those treated with vehicle. Conclusion Therapy with AAT suppresses the acute rejection after LuTx in a mouse model. The beneficial effects seem to involve anti-protease and immunomodulatory activities of AAT.

Indoleamine 2,3-Dioxygenase Cannot Inhibit Chlamydia trachomatis Growth in HL-60 Human Neutrophil Granulocytes

AimsNeutrophil granulocytes are the major cells involved in Chlamydia trachomatis (C. trachomatis)-mediated inflammation and histopathology. A key protein in human intracellular antichlamydial defense is the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) which limits the growth of the tryptophan auxotroph Chlamydia. Despite its importance, the role of IDO in the intracellular defense against Chlamydia in neutrophils is not well characterized.MethodsGlobal gene expression screen was used to evaluate the effect of C. trachomatis serovar D infection on the transcriptome of human neutrophil granulocytes. Tryptophan metabolite concentrations in the Chlamydia-infected and/or interferon-gamma (IFNG)-treated neutrophils were measured by ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS).ResultsOur results indicate that the C. trachomatis infection had a major impact on neutrophil gene expression, inducing 1,295 genes and repressing 1,510 genes. A bioinformatics analysis revealed that important factors involved in the induction of neutrophil gene expression were the interferon-related transcription factors such as IRF1-5, IRF7-9, STAT2, ICSB, and ISGF3. One of the upregulated genes was ido1, a known infection- and interferon-induced host gene. The tryptophan-degrading activity of IDO1 was not induced significantly by Chlamydia infection alone, but the addition of IFNG greatly increased its activity. Despite the significant IDO activity in IFNG-treated cells, C. trachomatis growth was not affected by IFNG. This result was in contrast to what we observed in HeLa human cervical epithelial cells, where the IFNG-mediated inhibition of C. trachomatis growth was significant and the IFNG-induced IDO activity correlated with growth inhibition.ConclusionsIDO activity was not able to inhibit chlamydial growth in human neutrophils. Whether the IDO activity was not high enough for inhibition or other chlamydial growth-promoting host mechanisms were induced in the infected and interferon-treated neutrophils needs to be further investigated.

Induced Pluripotent Stem Cell-Derived Neutrophil Granulocytes - Flow Cytometry Analysis Reveals Distinct Subpopulations

Severe congenital neutropenia (SCN) comprises a spectrum of monogenic disorders characterized by impaired differentiation and function of neutrophil granulocytes. Since animal models often do not fully recapitulate human SCN phenotypes and primary bone marrow samples of patients are scarce, alternative strategies are desirable to study the genetic causes and mechanisms. Induced pluripotent stem cells (iPSCs) can be differentiated into neutrophil granulocytes, thereby presenting an excellent tool to study hematopoiesis and especially neutrophil differentiation in health and disease in vitro. Recently, neutrophil progenitors and mature neutrophils of murine and human bone marrow have been characterized by single-cell RNA sequencing, mass cytometry and flow cytometry and are now referred to as proNeu, preNeu, immature-Neu and mature-Neu, at least in part reflecting conventional morphological classification of myeloblasts, promyelocytes, myelocytes/metamyelocytes and band and segmented neutrophils, respectively. Here we developed a flow cytometry antibody panel and gating strategy, which robustly identified distinct myeloid subsets in iPS-derived neutrophils. We adopted a differentiation protocol, which consists of feeder- and serum-free differentiation of iPS cells by mesoderm induction and patterning, followed by lineage progression through hemogenic endothelium to hematopoietic progenitors and finally mature neutrophil granulocytes. Floating cells arise, which can be harvested continuously and analyzed by flow cytometry. Based on expression of cell surface molecules, we defined four subpopulations: After selecting for single, live, CD45 + and CD14 - cells, the different progenitor stages were first defined by their expression of CD117 and CD49d. CD117 midCD49d high cells were further stratified into SSC lowCD34 + cells and SSC highCD34 - cells, representing myeloblasts (proNeu1) and promyelocytes (proNeu2/preNeu), respectively. These cells progressed to CD117 -CD49d mid and were CD11b +CD101 +, which defines myelocytes/metamyelocytes (immature-Neus). CD117 -CD49d low cells were CD11b +CD101 + and expressed additionally CD16, resembling band/segmented neutrophils (mature-Neus). Additionally, these iPS-derived cells progressively expressed CD35, which is also a maturation marker of human myeloid cells in vivo. May-Grünwald-Giemsa staining of these four subpopulations (CD117 midCD49d highSSC lowCD34 +, CD117 mid CD49d highSSC highCD34 -, CD117 - CD49d midand CD117 - CD49d low) revealed homogenous populations of sorted cells, morphologically resembling myeloblasts, promyelocytes, myelocytes/metamyelocytes and band/segmented neutrophils, respectively. Ongoing studies in our lab make use of this model to a) validate the functional significance of rare genetic variants and b) further assess transcriptomic and proteomic changes on a single cell level. Thus, we provide a promising tool to study neutrophil differentiation in health and disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The role of neutrophils in the pathogenesis of ischemic stroke

Background. Immune responses and inflammation play an important role in the pathogenesis of ischemic stroke.Aim. To analyze the involvement of neutrophils in the pathogenesis of ischemic stroke.Results. Data on the contribution of neutrophil granulocytes to the development of local sterile inflammation and secondary brain injury in acute ischemic stroke were summarized. Mechanisms of neutrophil influence on thrombosis, neutrophil extracellular trap formation (NETosis), protease release, and direct interaction with platelets with subsequent formation of platelet-leukocyte aggregates were discussed. Available information on the effectiveness of reperfusion therapy and an association of changes in neutrophil activity with development of infectious complications of stroke were presented. In addition, research data were presented on the contribution of neutrophils to atherogenesis, which is one of the most important etiological factors in ischemic stroke. The review showed that the contribution of neutrophils to the pathogenesis of ischemic stroke is associated with implementation of their secretory, regulatory, and phagocytic functions, as well as with NETosis.Conclusion. It was shown that neutrophils are involved in the pathogenesis of ischemic stroke and modulate a response to treatment.

Kynurenic Acid and Its Analogue SZR-72 Ameliorate the Severity of Experimental Acute Necrotizing Pancreatitis

The pathophysiology of acute pancreatitis (AP) is not well understood, and the disease does not have specific therapy. Tryptophan metabolite L-kynurenic acid (KYNA) and its synthetic analogue SZR-72 are antagonists of the N-methyl-D-aspartate receptor (NMDAR) and have immune modulatory roles in several inflammatory diseases. Our aims were to investigate the effects of KYNA and SZR-72 on experimental AP and to reveal their possible mode of action. AP was induced by intraperitoneal (i.p.) injection of L-ornithine-HCl (LO) in SPRD rats. Animals were pretreated with 75-300 mg/kg KYNA or SZR-72. Control animals were injected with physiological saline instead of LO, KYNA and/or SZR-72. Laboratory and histological parameters, as well as pancreatic and systemic circulation were measured to evaluate AP severity. Pancreatic heat shock protein-72 and IL-1β were measured by western blot and ELISA, respectively. Pancreatic expression of NMDAR1 was investigated by RT-PCR and immunohistochemistry. Viability of isolated pancreatic acinar cells in response to LO, KYNA, SZR-72 and/or NMDA administration was assessed by propidium-iodide assay. The effects of LO and/or SZR-72 on neutrophil granulocyte function was also studied. Almost all investigated laboratory and histological parameters of AP were significantly reduced by administration of 300 mg/kg KYNA or SZR-72, whereas the 150 mg/kg or 75 mg/kg doses were less or not effective, respectively. The decreased pancreatic microcirculation was also improved in the AP groups treated with 300 mg/kg KYNA or SZR-72. Interestingly, pancreatic heat shock protein-72 expression was significantly increased by administration of SZR-72, KYNA and/or LO. mRNA and protein expression of NMDAR1 was detected in pancreatic tissue. LO treatment caused acinar cell toxicity which was reversed by 250 µM KYNA or SZR-72. Treatment of acini with NMDA (25, 250, 2000 µM) did not influence the effects of KYNA or SZR-72. Moreover, SZR-72 reduced LO-induced H2O2 production of neutrophil granulocytes. KYNA and SZR-72 have dose-dependent protective effects on LO-induced AP or acinar toxicity which seem to be independent of pancreatic NMDA receptors. Furthermore, SZR-72 treatment suppressed AP-induced activation of neutrophil granulocytes. This study suggests that administration of KYNA and its derivative could be beneficial in AP.

A Saccharomyces cerevisiae Fermentation Product (Olimond BB) Alters the Early Response after Influenza Vaccination in Racehorses

Saccharomyces cerevisiae (S. cerevisiae) fermentation products (SCFP) are used in animal husbandry as pre- and postbiotic feed supplements. A variety of immunomodulatory effects are noted in many species. The purpose of this study was to test the hypothesis that horses fed with SCFP containing feed additive Olimond BB display a modulated early immune response after influenza vaccination. Six horses received Olimond BB pellets (OLI) and five horses were fed placebo pellets (PLA) for 56 days. On day 40 all horses were vaccinated with a recombinant influenza A/equi-2 vaccine. At the day of vaccination, the groups did not differ in the composition of leukocyte subpopulations and reticulocytes. Twenty-four hours after vaccination total leukocyte counts and numbers of CD4+ T-cells significantly increased in both groups. In PLA horses, the numbers of neutrophil granulocytes significantly increased and numbers of CD8+ T-cells decreased, whereas the numbers of these cell types remained unchanged in OLI horses. Only OLI horses displayed a significant increase in reticulocyte percentages after vaccination. The numbers of lymphocytes, monocytes, CD21+ B-cells, and serum amyloid A levels remained unaffected in both groups after vaccination. Sixteen days after vaccination, PLA and OLI horses differed significantly in their enhanced ELISA IgG titres against Newmarket and Florida Clade 1 influenza strains. The observed differences after vaccination suggest that feed supplementation with Olimond BB leads to modulated early immune responses after influenza vaccination, which may also affect the memory responses after booster vaccination.

Investigating the Role of Ly6G+ Neutrophils in Incisional and Inflammatory Pain by Multidimensional Pain-Related Behavioral Assessments: Bridging the Translational Gap

In recent years, preclinical pain research has failed to develop genuinely new analgesics for clinical use. This fact is reflected by a high number of patients, limited drug efficacy accompanied by side effects, and a long-term opioid intake. Two main aspects have been addressed, which hinder translation: the use of non-relevant pain models and a mismatch between pain-related outcomes in preclinical and clinical studies. Conversely, disease-specific pain models that mirror more closely the clinical situation and multidimensional behavioral outcome measures that objectively and reproducibly assess relevant pain-related symptoms in a preclinical setting could improve translation. Mechanistically, a matter of debate is the role of Ly6G+ neutrophil granulocytes (NGs) for pain. NGs are essential to eliminate pathogens and promote the wound healing process. For this purpose, there is a need to release various pro- and anti-inflammatory mediators, some of which could ameliorate or enhance pain. However, the contribution of NGs to different pain entities is contradictory for reflex-based tests, and completely unknown in the context of non-evoked pain (NEP) and movement-evoked pain (MEP). First, we combined withdrawal reflex-based assays with novel video-based assessments for NEP- and MEP-related behavior in two mouse pain models. The pain models utilized in this study were incision (INC) and pathogen/adjuvant-induced inflammation (CFA), translating well to postsurgical and inflammatory pain entities. Second, we depleted NGs and applied a set of behavioral assessments to investigate the role of NG migration in different pain modalities. Our comprehensive behavioral approach identified pain-related behaviors in mice that resemble (NEP) or differentiate (MEP) behavioral trajectories in comparison to mechanical and heat hypersensitivity, thereby indicating modality-dependent mechanisms. Further, we show that injury-induced accumulation of NGs minimally affects pain-related behaviors in both pain models. In conclusion, we report a novel assessment to detect NEP in mice after unilateral injuries using a more unbiased approach. Additionally, we are capable of detecting an antalgic gait for both pain entities with unique trajectories. The different trajectories between MEP and other pain modalities suggest that the underlying mechanisms differ. We further conclude that NGs play a subordinate role in pain-related behaviors in incisional and inflammatory pain.