Neutrophil Oxidative Burst Research Articles

Impact of Nitric Oxide on Polymorphonuclear Neutrophils' Function.

There is increasing evidence that nitric oxide (nitrogen monoxide, NO) significantly influences immune cellular responses, including those from polymorphonuclear leukocytes (PMNs). The aim of this study was to examine a possible effect of NO on PMNs' function (chemotaxis, production of reactive oxygen species (ROS), and NETosis) using live cell imaging. Moreover, we investigated PMN surface epitope and neutrophil oxidative burst under the influence of NO by flow cytometric analysis. Whole blood samples were obtained from healthy volunteers, and PMNs were isolated by density centrifugation. Live cell imaging using type I collagen matrix in µSlide IBIDI chemotaxis chambers was conducted in order to observe N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP)-stimulated PMN chemotaxis, ROS production, and NETosis. In the test group, NO was continuously redirected into the climate chamber of the microscope, so the chemotaxis chambers were surrounded by NO. The same experimental setup without NO served as a control. In addition, isolated PMNs were incubated with nitrogen monoxide (NO) or without (the control). Subsequently, flow cytometry was used to analyze neutrophil antigen expression and oxidative burst. Our live cell imaging results demonstrated a migration-promoting effect of NO on PMNs. We observed that in the case of prior stimulation by fMLP, NO has no effect on the time course of neutrophil ROS production and NET release. However, flow cytometric analyses demonstrated an increase in ROS production after pretreatment with NO. No NO-dependent differences for the expression of CD11b, CD62L, or CD66b could be observed. We were able to demonstrate a distinct effect of NO on PMNs' function. The complex interaction between NO and PMNs remains a major research focus, as the exact mechanisms and additional influencing factors remain elusive. Future studies should explore how varying NO concentrations and the timing of NO exposure relative to PMN activation affect its influence.

Neutrophil Oxidative Burst Profile Is Related to a Satisfactory Response to Itraconazole and Clinical Cure in Feline Sporotrichosis.

Despite the central role of cats in the transmission and amplification of Sporothrix, studies regarding immune response in feline sporotrichosis are scarce. In cats with sporotrichosis, neutrophil-rich lesions are usually associated to good general condition and lower fungal burden. However, the role of neutrophils in anti-Sporothrix immunity has been little explored in cats. Thus, the aim of this study was to evaluate the neutrophil oxidative burst in the blood of cats with sporotrichosis. Cats with sporotrichosis included in the study were treated with itraconazole (ITZ) alone or combined with potassium iodide (KI). The neutrophil oxidative burst was evaluated through a flow-cytometry-based assay using dihydrorhodamine 123 (background) and stimulation with Zymosan and heat-killed Sporothrix yeasts. The cure rate was 50.0% in cats under treatment with ITZ monotherapy and 90.9% in cats treated with ITZ + KI (p = 0.014), endorsing the combination therapy as an excellent alternative for the treatment of feline sporotrichosis. Higher percentages of Sporothrix-stimulated neutrophils were associated with good general condition (p = 0.003). Higher percentages of Sporothrix- (p = 0.05) and Zymosan-activated (p = 0.014) neutrophils before and early in the treatment were related to clinical cure in ITZ-treated cats. The correlation between oxidative burst and successful use of KI could not be properly assessed given the low number of failures (n = 2) in this treatment group. Nasal mucosa involvement, typically linked to treatment failure, was related to lower percentages of activated neutrophils in the background at the treatment outcome (p = 0.02). Our results suggest a beneficial role of neutrophils in feline sporotrichosis and a positive correlation between neutrophil activation and the cure process in ITZ-treated cats.

Neutrophil NADPH oxidase regulation by oxidative phosphorylation and accessory glucose metabolic pathways

Neutrophils produce reactive oxygen species (ROS) to extinguish pathogens and regulate intracellular signaling. The major producer of ROS in neutrophils is the NADPH oxidase, which is fueled by NADPH generated via the oxidative pentose phosphate pathway. It remains unclear how other accessory pathways of glucose metabolism and mitochondrial activity influence the oxidative burst in neutrophils. The respiratory burst in bone marrow-derived neutrophils was stimulated with phorbol 12-myristate 13-acetate (PMA) in the presence or absence of an inhibitor of glycogen breakdown (glycogen phosphorylase inhibitor; GPi), inhibitors of mitochondrial respiration (antimycin A and rotenone), or an inhibitor of serine biosynthesis (NCT503). Respiratory burst was also measured under conditions of limited glucose and glutamine. ROS production was measured via extracellular flux analysis or via dihydrorhodamine fluorescence. In both male and female mouse neutrophils, inhibition of glycogen breakdown by GPi delayed initiation of the oxidative burst in response to PMA (n = 6, p<0.05, Student’s T-test and Repeated Measure’s ANOVA). Inhibition of mitochondrial oxidative phosphorylation or inhibition of the serine biosynthetic pathway sustained ROS production in PMA-stimulated neutrophils (n = 6, p<0.05, Student’s T-test). Removal of glucose and glutamine showed a truncated oxidative burst (n = 6, p<0.05, Student’s T-test). Ultimately, these findings suggest that glycogen breakdown fuels the initial activation of the oxidative burst, while mitochondrial activity and the serine biosynthesis pathway control the magnitude and duration of neutrophil ROS production. Furthermore, utilization of exogenous glucose and glutamine are necessary for the entire oxidative burst. Future studies will continue to illustrate the mechanisms of neutrophil metabolic plasticity as well as connect their significance in the context of wound healing, exercise, and chronic inflammatory diseases. NIH: R01HL141191, NIH: P01 HL078825. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Innate and adaptive immune responses in subjects with CPA secondary to post-pulmonary tuberculosis lung abnormalities.

Post-tuberculosis lung abnormality (PTLA) is the most common risk factor for chronic pulmonary aspergillosis (CPA), and 14%-25% of the subjects with PTLA develop CPA. The pathogenesis and the host immune response in subjects with PTLA who develop CPA need to be better understood. We prospectively compared the innate and adaptive immune responses mounted by patients of PTLA with or without CPA (controls). We studied the neutrophil oxidative burst (by dihydrorhodamine 123 test), classic (serum C3 and C4 levels) and alternative (mannose-binding lectin [MBL] protein levels) complement pathway, serum immunoglobulins (IgG, IgM and IgA), B and T lymphocytes and their subsets in subjects with PTLA with or without CPA. We included 111 subjects (58 CPA and 53 controls) in the current study. The mean ± SD age of the study population was 42.6 ± 15.7 years. The cases and controls were matched for age, gender distribution and body weight. Subjects with CPA had impaired neutrophil oxidative burst, lower memory T lymphocytes and impaired Th-1 immune response (lower Th-1 lymphocytes) than controls. We found no significant difference between the two groups in the serum complement levels, MBL levels, B-cell subsets and other T lymphocyte subsets. Subjects with CPA secondary to PTLA have impaired neutrophil oxidative burst and a lower Th-1 response than controls.

Proteome and Dihydrorhodamine Profiling of Bronchoalveolar Lavage in Patients with Chronic Pulmonary Aspergillosis.

Neutrophil and (alveolar) macrophage immunity is considered crucial for eliminating Aspergillus fumigatus. Data derived from bronchoalveloar lavage (BAL) characterizing the human immuno-pulmonary response to Aspergillus fumigatus are non-existent. To obtain a comprehensive picture of the immune pathways involved in chronic pulmonary aspergillosis (CPA), we performed proteome analysis on AL of 9 CPA patients and 17 patients with interstitial lung disease (ILD). The dihydrorhodamine (DHR) test was also performed on BAL and blood neutrophils from CPA patients and compared to blood neutrophils from healthy controls (HCs). BAL from CPA patients primarily contained neutrophils, while ILD BAL was also characterized by a large fraction of lymphocytes; these differences likely reflecting the different immunological etiologies underlying the two disorders. BAL and blood neutrophils from CPA patients displayed the same oxidative burst capacity as HC blood neutrophils. Hence, immune evasion by Aspergillus involves other mechanisms than impaired neutrophil oxidative burst capacity per se. CPA BAL was enriched by proteins associated with innate immunity, as well as, more specifically, with neutrophil degranulation, Toll-like receptor 4 signaling, and neutrophil-mediated iron chelation. Our data provide the first comprehensive target organ-derived immune data on the human pulmonary immune response to Aspergillus fumigatus.

Impact of bronchoalveolar lavage from influenza A virus diseased pigs on neutrophil functions and growth of co-infecting pathogenic bacteria.

Influenza A viruses (IAVs) infect the respiratory tract of mainly humans, poultry, and pigs. Co-infections with pathogenic lung bacteria are a common event and contribute to the severity of disease progression. Neutrophils are a major cell type of the innate immune system and are rapidly recruited to the site of infection. They have several effector functions to fight invading pathogens such as the secretion of reactive oxygen species (ROS) or the release of neutrophil extracellular traps (NETs). NETs are known to promote the growth of Pasteurellaceae bacteria, especially if degraded by nucleases. In this study, bronchoalveolar lavage fluid (BALF) from 45 field-infected pigs was analyzed for 1) NET markers, 2) influence on growth of lung bacteria, and 3) impact on neutrophil functions. BALF samples from 21 IAV-positive pigs and 24 lung diseased but IAV-negative pigs were compared. Here, we show that neutrophils in the lungs of IAV-positive pigs release vesicular NETs. Several NET markers were increased in the BALF of IAV-positive pigs compared with the BALF from IAV-negative pigs. The amount of NET markers positively correlated with the viral load of the IAV infection. Interestingly, the BALF of IAV-positive pigs enhanced the growth of bacteria belonging to the family of Pasteurellaceae as potential coinfecting bacteria. These effects were weaker with the BALF derived from IAV-negative pigs with other lung infections. The intensity of oxidative burst in neutrophils was significantly decreased by BALF from IAVpositive pigs, indicating impaired antimicrobial activity of neutrophils. Finally, the lung milieu reflected by IAV-positive BALF does not enable neutrophils to kill Actinobacillus pleuropneumoniae but rather enhances its growth. In summary, our data show that an IAV infection is affecting neutrophil functions, in particular the release of NETs and ROS. Furthermore, IAV infection seems to provide growth-enhancing factors for especially coinfecting Pasteurellaceae and reduces the killing efficiency of neutrophils.

Measurement of the Neutrophils Count and Oxidative Burst in Neutrophils of Patients with Sanjad Sakati Syndrome.

Sanjad Sakati Syndrome (SSS) is categorized as a neuroendocrine-related disease due to disorders of the nervous and hormonal systems. Since hormonal changes in these patients may affect the nature and function of the immune system. Thus, in this study, cell count and phagocytotic function of neutrophils were evaluated which may be influenced by changes in the hormonal rate and growth factors. In this study, the neutrophil count value and the oxidative burst were evaluated in six patients diagnosed with SSS and six healthy individuals. There was a significant reduction in the neutrophil count observed in SSS patients compared to healthy controls (37.41±7.93 percent vs. 66.5±6.8 percent). However, there was no significant difference in neutrophil oxidative index between patients with SSS and control subjects (172.33±55.08 vs. 217.00±77.38). We concluded that in patients with SSS, the phagocytic activity of neutrophils was not affected by hormonal changes, while the number of neutrophils and neutrophil-to-lymphocyte ratio (NLR) index were decreased.

A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen.

Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils.

Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice.

Nonalcoholic steatohepatitis (NASH) is an advanced stage of fatty liver disease characterized by liver damage, inflammation, and fibrosis. Although neutrophil infiltration is consistently observed in the livers of patients with NASH, the precise role of neutrophil-recruiting chemokines and infiltrating neutrophils in NASH pathogenesis remains poorly understood. Here, we aimed to elucidate the role of neutrophil infiltration in the transition from fatty liver to NASH by examining hepatic overexpression of interleukin-8 (IL8), a major chemokine responsible for neutrophil recruitment in humans. Mice fed a high-fat diet (HFD) for 3 months developed fatty liver without concurrent liver damage, inflammation, and fibrosis. Subsequent infection with an adenovirus overexpressing human IL8 for an additional 2 weeks increased IL8 levels, neutrophil infiltration, and liver injury in mice. Mechanistically, IL8-induced liver injury was associated with the upregulation of components of the NADPH oxidase 2 complex, which participate in neutrophil oxidative burst. IL8-driven neutrophil infiltration promoted macrophage aggregate formation and upregulated the expression of chemokines and inflammatory cytokines. Notably, IL8 overexpression amplified factors associated with fibrosis, including collagen deposition and hepatic stellate cell activation, in HFD-fed mice. Collectively, hepatic overexpression of human IL8 promotes neutrophil infiltration and fatty liver progression to NASH in HFD-fed mice.

Biocompatibility of colloidal polypyrrole

The excellent electroconductive properties of polypyrrole (PPy) predetermine its use as cell-instructive or biosensing biomaterial. PPy has potential for application in a physiological environment, and here the cytotoxicity, antioxidant capacity, modulation of neutrophil oxidative burst, antibacterial activity of colloidal PPy stabilized with polyvinylpyrrolidone (PVP), and long-term stability under physiological conditions are reported. Besides, long-term stability under physiological conditions is also provided. The formation of PPy/PVP colloid was proven by UV-Vis and IR spectroscopy, and its morphology and particle size were detected. The results of cytotoxicity studies show that the safe PPy concentration in colloidal particles for biomedical usage is ≤ 200 µg mL−1. In these concentrations, the PPy/PVP also exhibits excellent antioxidant properties and modulates the immune response. The minimal inhibitory concentration of 234 µg mL−1 was determined against Staphylococcus aureus and Escherichia coli, respectively. The low cytotoxicity and bioactivity of the colloidal PPy/PVP demonstrate their long-term potential for a variety of applications

A Comparative Study of the Inhibitory Effect of Some Flavonoids and a Conjugate of Taxifolin with Glyoxylic Acid on the Oxidative Burst of Neutrophils.

During the storage, processing, and digestion of flavonoid-rich foods and beverages, a condensation of flavonoids with toxic carbonyl compounds occurs. The effect of the resulting products on cells remains largely unknown. The aim of the present study was to evaluate the effects of quercetin, taxifolin, catechin, eriodictyol, hesperetin, naringenin, and a condensation product of taxifolin with glyoxylic acid on the oxidative burst of neutrophils. It was found that the flavonoids and the condensation product inhibited the total production of ROS. Flavonoids decreased both the intra and extracellular ROS production. The condensation product had no effect on intracellular ROS production but effectively inhibited the extracellular production of ROS. Thus, the condensation of flavonoids with toxic carbonyl compounds may lead to the formation of compounds exhibiting potent inhibitory effects on the oxidative burst of neutrophils. The data also suggest that, during these reactions, the influence of a fraction of flavonoids and their polyphenolic derivatives on cellular functions may change. On the whole, the results of the study provide a better understanding of the effects of polyphenols on human health. In addition, these results reveal the structure-activity relationship of these polyphenols and may be useful in a search for new therapeutic agents against diseases associated with oxidative stress.

The chemorepellent, SLIT2, bolsters innate immunity against Staphylococcus aureus.

Neutrophils are essential for host defense against Staphylococcus aureus (S. aureus). The neuro-repellent, SLIT2, potently inhibits neutrophil chemotaxis, and might, therefore, be expected to impair antibacterial responses. We report here that, unexpectedly, neutrophils exposed to the N-terminal SLIT2 (N-SLIT2) fragment kill extracellular S. aureus more efficiently. N-SLIT2 amplifies reactive oxygen species production in response to the bacteria by activating p38 mitogen-activated protein kinase that in turn phosphorylates NCF1, an essential subunit of the NADPH oxidase complex. N-SLIT2 also enhances the exocytosis of neutrophil secondary granules. In a murine model of S. aureus skin and soft tissue infection (SSTI), local SLIT2 levels fall initially but increase subsequently, peaking at 3 days after infection. Of note, the neutralization of endogenous SLIT2 worsens SSTI. Temporal fluctuations in local SLIT2 levels may promote neutrophil recruitment and retention at the infection site and hasten bacterial clearance by augmenting neutrophil oxidative burst and degranulation. Collectively, these actions of SLIT2 coordinate innate immune responses to limit susceptibility to S. aureus.

Allogeneic HSCT for Symptomatic Female X-linked Chronic Granulomatous Disease Carriers

X-linked chronic granulomatous disease (XL-CGD) is an inherited disorder of superoxide production, causing failure to generate the oxidative burst in phagocytes. It is characterized by invasive bacterial and fungal infections, inflammation, and chronic autoimmune disease. While XL-CGD carriers were previously assumed to be healthy, a range of clinical manifestations with significant morbidity have recently been described in a subgroup of carriers with impaired neutrophil oxidative burst due to skewed lyonization. Allogeneic hematopoietic stem cell transplantation (HSCT) is the standard curative treatment for CGD but has rarely been reported in individual symptomatic carriers to date. We undertook a retrospective international survey of outcome of HSCT for symptomatic XL-CGD carriers. Seven symptomatic female XL-CGD carriers aged 1–56 years underwent HSCT in four centers, indicated for severe and recurrent infection, colitis, and autoimmunity. Two patients died from transplant-related complications, following donor engraftment and restoration of oxidative burst. All surviving patients demonstrated resolution of their neutrophil oxidative burst defect with concordant reduction in infection and inflammatory symptoms and freedom from further immunosuppressive therapy. In conclusion, allogeneic HSCT may cure the phagocyte defect in symptomatic XL-CGD carriers and improve their recurrent and disabling infective and inflammatory symptoms but risks transplant-related complications.

Cytomegalovirus durably primes neutrophil oxidative burst.

Cytomegalovirus (CMV) is a ubiquitous herpes virus that infects most humans, thereafter persisting lifelong in tissues of the host. It is a known pathogen in immunosuppressed patients, but its impact on immunocompetent hosts remains less understood. Recent data have shown that CMV leaves a significant and long-lasting imprint in host immunity that may confer some protection against subsequent bacterial infection. Such innate immune activation may come at a cost, however, with potential to cause immunopathology. Neutrophils are central to many models of immunopathology, and while acute CMV infection is known to influence neutrophil biology, the impact of chronic CMV infection on neutrophil function remains unreported. Using our murine model of CMV infection and latency, we show that chronic CMV causes persistent enhancement of neutrophil oxidative burst well after resolution of acute infection. Moreover, this in vivo priming of marrow neutrophils is associated with enhanced formyl peptide receptor expression, and ultimately constitutive c-Jun N-terminal kinase phosphorylation and enhanced CD14 expression in/on circulating neutrophils. Finally, we show that neutrophil priming is dependent on viral load, suggesting that naturally infected human hosts will show variability in CMV-related neutrophil priming. Altogether, these findings represent a previously unrecognized and potentially important impact of chronic CMV infection on neutrophil responsiveness in immunocompetent hosts.

Unique ulcerative autoinflammatory disease mistaken for factitious disorder

Autoinflammatory diseases (AID) are disorders of inappropriate innate immune system activation. Twenty-four percent of patients with AID do not have a well-defined clinical picture or pathogenic mutation and are characterized as having undefined AID. Cutaneous ulcers have been described but not outside of distinct histologic and clinical disorders. We present a unique case of undefined adult-onset AID with ulcers mistaken for a factitious disorder.A 32 year-old female with borderline personality disorder, depression, and anxiety developed recurrent cutaneous MRSA abscesses and recurrent ulcers of the extremities and scalp associated with alopecia. The ulcers were minimally-responsive to IV antibiotics, isotretinoin, oral dapsone, intralesional triamcinolone, and adalimumab. Flares of the skin disease were associated with fevers and elevated inflammatory markers.Scalp ulcer biopsies revealed numerous pigmented hair casts with trichomalacia and perifollicular lymphocytic infiltrate. One hair follicle contained bacterial cocci associated with a perifollicular neutrophilic infiltrate. These findings along with the patient's psychiatric history led to a diagnosis of trichotillomania from multiple dermatology and psychiatry consultations. The patient denied that she was inducing the lesions and sought another psychiatric consultation, who felt this was not a factitious disease. She was referred to immunology for evaluation of a possible IEI. Laboratory evaluation revealed mild hypogammaglobulinemia with IgG of 663 (768–1632) mg/dL, normal antibody response to recall antigens, but absent response to neo-antigen (Salmonella typhi). Peripheral lymphocyte phenotyping was normal except for a relative decrease in the number of memory B-cells with CD27+IgD+% of 6.9 (13.4–21.4) and CD27+IgD-% of 2.5 (9.2–18.9). Neutrophil oxidative burst was normal. Whole exome sequencing did not identify variants in genes associated with her phenotype.The patient was partially responsive to IVIG but highly responsive to high-dose intravenous methylprednisolone. She was subsequently diagnosed with undefined AID based on Eurofever registry criteria matching her clinical course and had near complete resolution of skin lesions and alopecia with the addition of anakinra.Clinicians must be vigilant for atypical presentations of AID. Our patient had signs of undefined AID but had a delayed diagnosis due to her psychiatric disorders, to which her symptoms were attributed, and a previously undescribed presentation of AID.

Use of Interferon Gamma in Female Carriers of X-linked Chronic Granulomatous Disease

IntroductionChronic granulomatous disease (CGD) is characterized by increased frequency and severity of certain infections, autoimmune disease, and hyperinflammation. Heterozygous female carriers of X-linked CGD may experience similar symptoms. Guidance for management of female carriers is limited. We describe the use of interferon gamma (IFNγ) in X-linked CGD carriers. MethodsA multi-center retrospective chart review identified 4 carriers of X-linked CGD who received prophylaxis with IFNγ. Results of dihydrorhodamine (DHR) assay, infections and autoinflammatory features were documented and adverse events (AE) related to IFNγ were recorded. ResultsFour females (mean age 40 [range 0.5–71] years) received IFNγ (75–100 µg 3 times per week via subcutaneous injection) for a mean duration of 43 (range 16–63) months. Infections prior to the start of IFNγ included sepsis/bacteremia, pneumonia, skin and soft tissue infection, osteomyelitis, and gastroenteritis with CGD-associated pathogens including Serratia marscescens, Burkholderia cepacia, Nocardia spp, and methicillin-resistant Staphylococcus aureus (MRSA). Inflammatory complications (some in more than one subject) included dermatologic (pyoderma gangrenosum [n = 1], unspecified dermatitis [n = 2]), rheumatologic (arthritis [n = 1], Raynaud's syndrome [n = 1]), and gastrointestinal (inflammatory diarrhea [n = 1], Barrett's esophagus [n = 1]). Each carrier exhibited two populations of neutrophil oxidative burst. The populations expressing DHR were as follows: 0.08, 1, 3, and 10.8 +DHR%. Antibacterial prophylaxis was administered in all 4 subjects, and antifungal prophylaxis in 3 subjects. Pre-medications were not administered, and AE associated with IFNγ were mild or moderate in 3 of 4 subjects (one subject had no AE) and did not result in any treatment discontinuations. One patient discontinued prophylaxis with IFNγ after correction of CGD following hematopoietic stem cell transplant. The average length of follow-up since starting IFNγ was 38 months. While on IFNγ therapy, one patient developed atypical mycobacterial and cytomegalovirus pneumonia and MRSA chronic osteomyelitis thought related to poor compliance, while another developed Serratia olecranon abscess/. None developed autoimmune/inflammatory disease while on therapy. ConclusionsThis is the first report to our knowledge of IFNγ use for prophylaxis in carriers of X-linked CGD. IFNγ was well tolerated, and should be considered for prophylaxis in female carriers with very low neutrophil oxidative burst or frequent or severe CGD-associated infections.

Inborn Error of Immunity Presenting with Salmonella Pericarditis and Pericardial Effusion

Salmonella infections typically occur in disorders of innate immunity with impairment in phagocyte function and/or impaired signaling in the IL-12/interferon (IFN)-gamma axis. Pericarditis/pericardial effusion has previously been reported as a rare complication of Salmonella infection.An 8-year-old, previously healthy, fully immunized Hispanic female presented with 4 days of fever and 5 days of pain in her chest, abdomen, and left shoulder. Her medical and family history was unremarkable. Chest radiograph revealed cardiomegaly, and a transthoracic echocardiogram identified a large pericardial effusion. Labs showed leukocytosis (15 K/uL), neutrophilia (ANC 12.77 K/uL), elevated CRP (287 mg/dL), and elevated BNP (607 pg/mL). Exam showed mild distress, gallop on cardiac auscultation, diminished left breath sounds, cool extremities, and 1+ peripheral pulses. She underwent pericardiocentesis, and the fluid culture was positive for Salmonella alachua. She was treated with IV ceftriaxone for 8 days, then transitioned to trimethoprim/sulfamethoxazole (TMP/SMX) to complete a 30-day total antibiotic course.Inpatient immunologic evaluation revealed T cell lymphopenia (TCL) with decreased naïve CD4+ and CD8+ T cells. Increased CD4+ and CD8+ effector memory T cells (Tem) were also observed, along with increased CD8+ TEMRA, and certain subsets of CD4+/CD8+ central memory T cells lacking CD62L. HIV testing and neutrophil oxidative burst were negative. Given her significant T cell lymphopenia, TMP/SMX prophylaxis was continued after the treatment course, and fluconazole prophylaxis was initiated due to endemic coccidiomycosis.A targeted 429-gene panel for inborn errors of immunity revealed a heterozygous missense variant of uncertain significance in PIK3CD, = c.1013G>A, p.Arg338Gln. This variant is not in gnomAD for Hispanics and is present in 0.0009% of non-Finnish Europeans. Gain-of-function (GoF) PIK3CD mutations cause a combined immunodeficiency, with increased risk of infection from encapsulated organisms, such as salmonella. However, the patient’s immunophenotype is not consistent with GoF PIK3CD mutation. She had normal transitional B cells, lack of increased senescent T cells, and absence of lymphoproliferation.TCL is likely an immunologic risk factor for Salmonella pericarditis with pericardial effusion. Though the PI3CKD variant does not appear to be pathogenic, additional functional testing for this variant is in progress.

Primary immunodeficiency with autoimmunity, two facets of same coin?-A novel case with therapeutic dilemma

12 year female born of Grade 2 consanguineous marriage presented with history of painful swelling in the right axilla and neck for 2 months. She also had history of multiple joint pain involving small and large joints with early morning stiffness for 2 months. She had past history of treatment for tubercular cervical lymphadenitis (caseating granuloma in biopsy) at 4 years of age. She was immunized appropriate for her age. Her father and paternal aunt had psoriasis. On examination she had warm and tender subcutaneous swelling 2 × 2 cm in right axilla. Significant lymphadenopathy noted in right cervical and femoral region. Psoriatic plaque was noted over scalp and right retroauricular area.Musculoskeletal examination showed bilateral wrist, ankle arthritis and bilateral achilles tendinitis. On evaluation her hemoglobin 11 mg/dl, WBC count 19000 (N88%, L9%, M3%), platelet 665000 with neutrophilic leukocytosis and shift to left. Ultrasound of right axilla confirmed abscess. Incision and drainage of abscess was done. Pus culture grew S.aureus. She was diagnosed as psoriatic arthritis with axillary abscess and generalized significant lymphadenopathy. In view of abscess, generalized lymphadenopathy, leukocytosis and neutrophilia, PID workup was done. Her immunoglobulin levels were normal. Lymphocyte subset analysis showed reduced CD8+T lymphocytes. NBT activity was seen in 55% of stimulated neutrophils (90% in control sample). DHR assay (test done twice 2 weeks apart) showed markedly decreased Neutrophil oxidative burst. Cervical lymph node biopsy revealed granulomatous inflammation. CBNAAT of sample was negative for tuberculosis. Her genetic workup showed mutation for a novel missense variant c.908T>G in Exon 10 of NCF4 and carrier state in parents and her HLA typing was positive for HLACw6. She was diagnosed as chronic granulomatous disease with psoriatic arthritis. She received sulfamethoxazole/trimethoprim, itraconazole prophylaxis and treatment with methotrexate 7.5 mg/week for arthritis. Discussion1. CGD is associated with few autoimmune manifestations like colitis, arthritis etc. To the best of our knowledge, association with Psoriatic arthritis is not reported. 2. Treatment of our case has unique challenging issues as use of DMARDS for psoriatic arthritis may increase infection chance in preexisting CGD and use of steroids for granuloma may cause rebound flare of psoriasis.

Abnormal Dihydrorhodamine Result in a Patient with Diabetes Mellitus, Recurrent Candida Liver Abscesses: Diagnostic Possibilities

A 17-year-old female with type I diabetes mellitus (DM) and a history of recurrent candidal vaginal infections and liver abscesses was assessed fora potential neutrophil oxidative burst defect by DHR flow cytometry. The DHR result was abnormal with significantly decreased MFI (MFI = 9; ref. cutoff: >/ = 60) after stimulation with PMA though all of her neutrophils (99%; ref. cutoff >/ = 95%) showed respiratory burst. This DHR pattern has been previously observed in patients with complete myeloperoxidase deficiency (cMPO), p40phox deficiency and hypomorphic X-linked chronic granulomatous disease (CGD). Genetic evaluation of the genes commonly associated with CGD (CYBB, CYBA, NCF1, NCF2 and NCF4) was negative. It must be noted that many genetic laboratories either do not sequence the NCF1 gene or have limited sequencing to the most commonly observed pathogenic variant in exon 2. Most targeted genetic panels currently do not include MPO (myeloperoxidase) or CYBC1 (Eros). The clinical phenotype of Candida infection in the context of diabetes has been previously reported in 5% of cMPO deficiency. The patient underwent a partial hepatectomy and was initiated on an 8-month course of fluconazole. Three months later, there was a recurrence of hepatic abscesses (Figure 1), requiring treatment with a 12-week course of IV micafungin. Complete MPO deficiency can cause a false-positive result on the DHR assay but the pattern is different from that observed in typical XL- and autosomal recessive CGD. There are two ways of confirming cMPO deficiency, besides genetic testing: 1) staining for MPO in neutrophils in a peripheral smear, or 2) repeating the DHR assay and gating on eosinophils as eosinophil peroxidase can overcome the lack of MPO deficiency and demonstrate a normal oxidative burst. These studies are currently under investigation. In summary, cMPO deficiency should be included in the differential diagnosis of patients with diabetes and invasive Candida infections. Communication with the clinical laboratory is essential to identify the DHR pattern and obtain additional confirmatory testing. From a clinical perspective, cMPO-deficient patients do not require prophylactic antibiotics but they have a high rate of recurrence of fungal infections and require aggressive management with antifungal therapies. [Display omitted]

Lupus nephritis with clinical and laboratory features of humoral immune deficiency in a 29-year-old female with a novel NCF1 variant consistent with autosomal recessive chronic granulomatous disease

Chronic Granulomatous Disease (CGD) is a rare, inherited disorder of the innate immune system characterized by defects in the phagocyte NADPH oxidase complex. This results in poor phagocyte function causing frequent infections (bacterial and fungal) and chronic inflammation. Here we discuss a rare presentation of lupus nephritis and antibody deficiency in an adult female patient with a novel homozygous nonsense mutation in the NCF-1 gene causing autosomal recessive CGD.Our patient is a 29-year-old female with history of ITP, biopsy-confirmed lupus nephritis, systemic lupus erythematosus (SLE), idiopathic avascular necrosis of the hip, and previous diagnosis of common variable immune deficiency (CVID) treated with Immunoglobulin replacement therapy (IVIG). She presented to our clinic to establish care in the setting of persistent skin abscesses/cellulitis, poor wound healing, necrotic lymphadenitis, and recurrent urinary tract infections and pneumonia despite years of IVIG therapy. She had near-normal total IgG, absent IgA and IgE, and protective vaccine antibody titers (pneumococcus, diphtheria, and tetanus) prior to the start of IVIG. Immunophenotyping demonstrated quantitatively normal number of total B cells, absent CD27+ memory B cells, and normal T cell subsets. She had reassuring lymphocyte proliferation with mitogen stimulation and normal TLR function. Dihydrorhodamine (DHR) flow cytometric assay demonstrated abnormally low neutrophil oxidative burst activity. Gene sequencing revealed homozygous nonsense mutation in the NCF-1 gene (c.75-76del; pY26Hfs*26) and a pathogenic heterozygous variant in FANCM (c.3732-3735dup; p. Leu1246Asnfs*8). She was started on daily prophylactic trimethoprim-sulfamethoxazole and itraconazole. She has marked decrease in infections/hospitalizations and her lupus nephritis is well controlled with hydroxy-chloroquine. She is being referred to the NIH for HSCT evaluation. The majority of CGD cases in the United States are due to X-linked CGD (70%) and AR variants in NCF1 (20%). While rare, discoid lupus erythematosus and SLE have been documented with X-linked forms of CGD. Similarly, there are few case reports of antibody deficiency in patients with CGD. This is the first documented case of AR CGD with SLE/lupus nephritis and humoral immune defects. Additionally, we report a disease-causing, novel variant in the NCF1 gene. This case report adds to the existing literature on CGD and associated diseases.