José Antonio T. Albuquerque

Cellular renewal and improvement of local cell effector activity in peritoneal cavity in response to infectious stimuli.

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (MØ) subsets, namely small peritoneal MØ (SPM) and large peritoneal MØ (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for β-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-γ stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal MØ subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

Deficiency of the Human Complement Regulatory Protein Factor H Associated with Low Levels of Component C9

We identified a 4‐year‐old Brazilian boy from a family of Japanese descent and history of consanguinity, who suffered from severe recurrent pneumonia. He carries factor H (FH) deficiency associated with reduced levels of component C9 and low serum levels of C3 and factor B. His mother also presented low levels of these proteins and factor I, while his father and sister had only lower levels of FH. Western blot assays confirmed the complete absence of FH and FHL‐1 polypeptides in this patient. Sequencing of the proband’s FH cDNA revealed a homozygous G453A substitution, encoding an Arg127His change. His mother, father and sister are heterozygous for this substitution. Despite the absence of FH in the plasma, this protein was detected in the patient’s fibroblasts, suggesting that Arg127 may be important for FH secretion. Low concentrations of C9 were detected in the proband serum but no mutations in the patient’s C9 gene or promoter have been identified, suggesting that this is a consequence of uncontrolled complement activation and high C9 consumption.

Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study

Enteroinvasive Escherichia coli (EIEC) and Shigella spp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigella share many genetic and biochemical similarities, the illness caused by Shigella is more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneri with cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed that S. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg127His mutation in FH (FHR127H) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-γ were able to synthesize FHR127H, the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-γ secrete FH without retention in the ER. Moreover, the retention of FHR127H provoked enlargement of ER cisterns after treatment with IFN-γ. A similar ER retention was observed in Cos-7 cells expressing the mutant FHR127H protein. Despite this deficiency in secretion, we show that the FHR127H mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient’s fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein.

A novel mutation in CYBB gene in a patient with chronic colitis and recurrent pneumonia due to X-linked chronic granulomatous disease

To the Editor: We report the case of a 20-year-old Brazilian male born from nonconsanguineous parents (Figure 1A) andno family history of immunodeficiency. He presented with a history of recurrent pneumonias since age 1.5 years, lymphadepathy, and chronic colitis. At age 3, a lung biopsy revealed noncaseous granulomatous disease. At age 5, a nitroblue tetrazolium assay was abnormal. At age 9, the patient was diagnosed with asthma after serious episodes of dyspnea and wheezing. In the same year, he was hospitalized for vomiting and abdominal pain; the upper gastrointestinal (UGI) endoscopy revealed milky lesions in esophagus, stomach, and duodenum. At age 15, a new UGI endoscopy revealed esophageal stenosis. Interferon gamma therapy was started at age 16 (subcutaneous, 50 μg/m2 three times perweek) with positive response. At age 17, he was hospitalized with severe pneumonia and inflammatory infiltration in the thoracicwall and in the fifth costal arch (Figure 1B). The dihydrorhodamine-123 (DHR) assay performed by flow cytometry showed an impaired respiratory burst after cell stimulation with phorbol 12-myristate 13 acetate (PMA) with low stimulation index (SI: 1.90) of patients granulocytes compared to a healthy control (SI: 45.35; Figure 1C). The patients mother DHR assay showed a bimodal population (SI: 28.40). The luminol-dependent chemiluminescence assay confirmed the impaired respiratory burst of patients neutrophils under PMA stimulation (AUC: 5.58 × 105) compared to a healthy control (AUC: 5.79 × 107, Figure 1D). The mothers neutrophil respiratory burst was lower compared to the healthy control (AUC: 1.77×107; Figure 1D). Patients neutrophils showed a low cytochrome b558 expression compared to a healthy control and the mothers neutrophils showed a bimodal histogram (Figure 1E). The patients genetic analysis (Sanger sequencing) revealed a novel missense mutation in the exon 8 (c.951T > A; p.V295E) of the CYBB gene; as expected the patients mother was heterozygous for the mutation (Figure 1F). The patient here reported underwent HSCTwith a good outcome. Chronic granulomatous disease (CGD) is an uncommon inherited immunodeficiency characterized by defects of the respiratory burst leading to impairedmicrobicidal activity of phagocytes.1 Gastrointestinal (GI) complications in CGD patients can cause morbidity and a substantial impact on quality of life. It is estimated that 50% of patients with CGD present with GI infections.2–5 Our patient had recurrent colitis and abdominal pain during childhood leading to a diagnosis of inflammatory bowel disease at age 9. The DHR assay is commonly used to diagnose CGD patients. The DHR with PMA can be normal in patients with mutations in Rac2/NCF4 genes,6 however the DHR test performed with N-formyl-methionyl-leucyl-phenylalanine may reveal abnormalities compatible with CGD. Molecular mutational analysis of our patient showed a change of a valine (V) for a glutamic acid (E) at position 295 in the gp91phox protein, a not previously reported mutation. It affects the gp91phox protein in position V295E, which is closely related to E309K and K315del previously reported mutations.7 These mutations are associatedwith decreased expression levels of gp91phox and impaired oxidative burst.

A C126R de novo Mutation in CYBB Leads to X-linked Chronic Granulomatous Disease With Recurrent Pneumonia and BCGitis

Chronic granulomatous disease (CGD) is an innate immune deficiency of phagocytic cells caused by mutations that affect components of the NADPH oxidase system, with resulting impairment in reactive oxygen species production. Patients with CGD are susceptible to recurrent infections and hyperinflammatory responses. Mutations in CYBB lead to the X-linked form of CGD and are responsible for ~ 70% of cases. In this study, we report the case of a 2.5-year-old male patient with recurrent pneumonia and Bacillus Calmette-Guérin infection (BCGitis). As his first clinical manifestation, he presented with bullous impetigo at 18 days of age, which was followed by recurrent pneumonia and regional BCGitis. Genetic analysis revealed a de novo mutation in exon 5 of the CYBB gene: a single-nucleotide substitution, c.376T > C, leading to a C126R change.

The Role of AIRE in the Immunity Against Candida Albicans in a Model of Human Macrophages

Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a primary immunodeficiency caused by mutations in the autoimmune regulator gene (AIRE). Patients with AIRE mutations are susceptible to Candida albicans infection and present with autoimmune disorders. We previously demonstrated that cytoplasmic AIRE regulates the Syk-dependent Dectin-1 pathway. In this study, we further evaluated direct contact with fungal elements, synapse formation, and the response of macrophage-like THP-1 cells to C. albicans hyphae to determine the role of AIRE upon Dectin receptors function and signaling. We examined the fungal synapse (FS) formation in wild-type and AIRE-knockdown THP-1 cells differentiated to macrophages, as well as monocyte-derived macrophages from APECED patients. We evaluated Dectin-2 receptor signaling, phagocytosis, and cytokine secretion upon hyphal stimulation. AIRE co-localized with Dectin-2 and Syk at the FS upon hyphal stimulation of macrophage-like THP-1 cells. AIRE-knockdown macrophage-like THP-1 cells exhibited less Dectin-1 and Dectin-2 receptors accumulation, decreased signaling pathway activity at the FS, lower C. albicans phagocytosis, and less lysosome formation. Furthermore, IL-1β, IL-6, or TNF-α secretion by AIRE-knockdown macrophage-like THP-1 cells and AIRE-deficient patient macrophages was decreased compared to control cells. Our results suggest that AIRE modulates the FS formation and hyphal recognition and help to orchestrate an effective immune response against C. albicans.