Diana L. Bonilla

Autophagy Regulates Phagocytosis by Modulating the Expression of Scavenger Receptors

Autophagy and phagocytosis are conserved cellular functions involved in innate immunity. However, the nature of their interactions remains unclear. We evaluated the role of autophagy in regulating phagocytosis in macrophages from myeloid-specific autophagy-related gene 7-deficient (Atg7⁻/⁻) mice. Atg7⁻/⁻ macrophages exhibited higher bacterial uptake when infected with Mycobacterium tuberculosis (Mtb) or with M. tuberculosis var. bovis BCG (BCG). In addition, BCG-infected Atg7⁻/⁻ mice showed increased bacterial loads and exacerbated lung inflammatory responses. Atg7⁻/⁻ macrophages had increased expression of two class A scavenger receptors: macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor 1 (MSR1). The increase in scavenger receptors was caused by increased activity of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) transcription factor resulting from accumulated sequestosome 1 (SQSTM1 or p62) in Atg7⁻/⁻ macrophages. These insights increase our understanding of the host-pathogen relationship and suggest that therapeutic strategies should be designed to include modulation of both phagocytosis and autophagy.

Pregnancy enhances the innate immune response in experimental cutaneous leishmaniasis through hormone-modulated nitric oxide production

The maintenance of host defense during pregnancy may depend on heightened innate immunity. We evaluated the immune response of pregnant hamsters during early infection with Leishmania (Viannia) panamensis, a cause of American cutaneous leishmaniasis. At 7 days post‐infection, pregnant animals showed a lower parasite burden compared with nonpregnant controls at the cutaneous infection site (P=0.0098) and draining lymph node (P=0.02). Resident peritoneal macrophages and neutrophils from pregnant animals had enhanced Leishmania killing capacity compared with nonpregnant controls (P=0.018 each). This enhanced resistance during pregnancy was associated with increased expression of inducible NO synthase (iNOS) mRNA in lymph node cells (P=0.02) and higher NO production by neutrophils (P=0.0001). Macrophages from nonpregnant hamsters infected with L. panamensis released high amounts of NO upon estrogen exposure (P=0.05), and addition of the iNOS inhibitor L‐N6‐(1‐iminoethyl) lysine blocked the induction of NO production (P=0.02). Infected, nonpregnant females treated with estrogen showed a higher percentage of cells producing NO at the infection site than controls (P=0.001), which correlated with lower parasite burdens (P=0.036). Cultured macrophages or neutrophils from estrogen‐treated hamsters showed significantly increased NO production and Leishmania killing compared with untreated controls. iNOS was identified as the likely source of estrogen‐induced NO in primed and naïve macrophages, as increased transcription was evident by real‐time PCR. Thus, the innate defense against Leishmania infection is heightened during pregnancy, at least in part as a result of estrogen‐mediated up‐regulation of iNOS expression and NO production.

Autophagy Is Required for Neutrophil-Mediated Inflammation

Autophagy, an intracellular degradation and energy recycling mechanism, is emerging as an important regulator of immune responses. However, the role of autophagy in regulating neutrophil functions is not known. We investigated neutrophil biology using myeloid-specific autophagy-deficient mice and found that autophagy deficiency reduced neutrophil degranulation in vitro and in vivo. Mice with autophagy deficiency showed reduced severity of several neutrophil-mediated inflammatory and autoimmune disease models, including PMA-induced ear inflammation, LPS-induced breakdown of blood-brain barrier, and experimental autoimmune encephalomyelitis. NADPH oxidase-mediated reactive oxygen species generation was also reduced in autophagy-deficient neutrophils, and inhibition of NADPH oxidase reduced neutrophil degranulation, suggesting NADPH oxidase to be a player at the intersection of autophagy and degranulation. Overall, this study establishes autophagy as an important regulator of neutrophil functions and neutrophil-mediated inflammation in vivo.

Incorporation of a Dietary Omega 3 Fatty Acid Impairs Murine Macrophage Responses to Mycobacterium tuberculosis

Background Beside their health benefits, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) might impair host resistance to Mycobacterium tuberculosis (Mtb) by creating an immunosuppressive environment. We hypothesized that incorporation of n-3 PUFA suppresses activation of macrophage antimycobacterial responses and favors bacterial growth, in part, by modulating the IFNγ-mediated signaling pathway. Methodology/Principal Findings Murine macrophage-like J774A.1 cells were incubated with bovine serum albumin (BSA)-conjugated docosahexaenoic acid (DHA; 22:6n-3) or BSA alone, activated with recombinant IFNγ, and infected with a virulent strain (H37Rv) of M. tuberculosis. The fatty acid composition of macrophage membranes was modified significantly by DHA treatment. DHA-treated macrophages were less effective in controlling intracellular mycobacteria and showed impaired oxidative metabolism and reduced phagolysosome maturation. Incorporation of DHA resulted in defective macrophage activation, as characterized by reduced production of pro-inflammatory cytokines (TNFα, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNγ receptor with lipid rafts, without affecting surface expression of IFNγ receptor. Conclusions/Significance We conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNγ, by modulation of IFNγ receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to tuberculosis.

Transgenic Mice Enriched in Omega-3 Fatty Acids Are More Susceptible to Pulmonary Tuberculosis: Impaired Resistance to Tuberculosis in fat-1 Mice

BACKGROUND. Besides their health benefits, dietary omega-3 fatty acids (n-3 PUFAs) can impair host resistance to intracellular pathogens. Previously, we and others have showed that n-3 PUFA-treated macrophages poorly control Mycobacterium tuberculosis infection in vitro. METHODS. Wild-type and fat-1 transgenic mice were infected with virulent H37Rv M. tuberculosis via the aerosol route. We evaluated bacteriological and histopathological changes in lungs, as well as differences in activation and antimycobacterial capacity in primary macrophages ex vivo. RESULTS. fat-1 mice were more susceptible to tuberculosis, as demonstrated by higher bacterial loads and less robust inflammatory responses in lungs. Macrophages obtained from fat-1 mice were more readily infected with M. tuberculosis in vitro, compared with wild-type macrophages. This impaired bacterial control in cells from fat-1 mice correlated with reduced proinflammatory cytokine secretion, impaired oxidative metabolism, and diminished M. tuberculosis-lysotracker colocalization within phagosomes. CONCLUSIONS. We showed that endogenous production of n-3 PUFAs in fat-1 mice increases their susceptibility to tuberculosis, which could be explained in part by diminished activation and antimycobacterial responses in cells from fat-1 mice. These data suggest that n-3 PUFA-supplemented diets might have a detrimental effect on immunity to M. tuberculosis and raise concerns regarding the safety of omega-3 dietary supplementation in humans.

Regulation of IL-4 Receptor Signaling by STUB1 in Lung Inflammation

RATIONALE IL-4Rα, the common receptor component for IL-4 and IL-13, plays a critical role in IL-4- and IL-13-mediated signaling pathways that regulate airway inflammation and remodeling. However, the regulatory mechanisms underlying IL-4Rα turnover and its signal termination remain elusive. OBJECTIVES To evaluate the role of STUB1 (STIP1 homology and U-Box containing protein 1) in regulating IL-4R signaling in airway inflammation. METHODS The roles of STUB1 in IL-4Rα degradation and its signaling were investigated by immunoblot, immunoprecipitation, and flow cytometry. The involvement of STUB1 in airway inflammation was determined in vivo by measuring lung inflammatory cells infiltration, mucus production, serum lgE levels, and alveolar macrophage M2 activation in STUB1(-/-) mice. STUB1 expression was evaluated in airway epithelium of patients with asthma and lung tissues of subjects with chronic obstructive pulmonary disease. MEASUREMENTS AND MAIN RESULTS STUB1 interacted with IL-4Rα and targeted it for ubiquitination-mediated proteasomal degradation, terminating IL-4 or IL-13 signaling. STUB1 knockout cells showed increased levels of IL-4Rα and sustained STAT6 activation, whereas STUB1 overexpression reduced IL-4Rα levels. Mice deficient in STUB1 had spontaneous airway inflammation, alternative M2 activation of alveolar macrophage, and increased serum IgE. STUB1 levels were increased in airways of subjects with asthma or chronic obstructive pulmonary disease, suggesting that up-regulation of STUB1 might be an important feedback mechanism to dampen IL-4R signaling in airway inflammation. CONCLUSIONS Our study identified a previously uncharacterized role for STUB1 in regulating IL-4R signaling, which might provide a new strategy for attenuating airway inflammation.

Congenital Transmission of Experimental Leishmaniasis in a Hamster Model

Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring born to L. panamensis-infected hamsters and offspring born to L. donovani-infected hamsters than controls, and lymphoproliferation to Leishmania was more frequent in offspring born to L. panamensis-infected hamsters (17.4%, 11 of 63) than in offspring born to L. donovani-infected hamsters (8.5%, 3 of 35). After weaning, only offspring born to L. donovani-infected hamsters had lower weight gain (P < 0.001) and hematocrit levels (P = 0.0045) than controls. Challenge of offspring born to L. panamensis-infected hamsters with L. panamensis showed no differences in lesion evolution, and offspring born to L. donovani-infected hamsters were more susceptible to L. donovani challenge than controls. Consequently, prenatal exposure of hamsters to L. donovani significantly increased the mortality risk and susceptibility to secondary homologous infection.

Expression of interferon-γ and tumour necrosis factor-α messenger RNA does not correlate with protection in guinea pigs challenged with virulent Mycobacterium tuberculosis by the respiratory route

Cytokine messenger RNA (mRNA) expression was investigated in the spleen and lung digest cells of bacillus Calmette–Guérin (BCG)‐vaccinated and non‐vaccinated guinea pigs following low‐dose, pulmonary exposure to virulent Mycobacterium tuberculosis. After purified protein derivative (PPD) stimulation, the levels of lung cell interferon‐γ (IFN‐γ), tumour necrosis factor‐α (TNF‐α) and spleen cell interleukin‐12 (IL‐12) p40 mRNAs were significantly increased in the non‐vaccinated M. tuberculosis‐infected guinea pigs compared to the BCG‐vaccinated guinea pigs. In contrast, the expression of anti‐inflammatory transforming growth factor‐β and IL‐10 mRNAs was significantly enhanced in the spleens of BCG‐vaccinated animals. Despite the presence of protective cytokine mRNA expression, the non‐vaccinated guinea pigs had significantly higher lung and spleen bacterial burdens. In contrast, BCG‐vaccinated guinea pigs controlled the bacterial multiplication in their lungs and spleens, indicating that both protective as well as anti‐inflammatory cytokine responses are associated with a reduction in bacteria. In addition, lung digest cells from non‐vaccinated guinea pigs contained a significantly higher percentage of neutrophils, CD3+ and CD8+ T cells, while the percentage of macrophages was increased in the BCG‐vaccinated animals. Total and purified lung digest T cells co‐cultured with lung macrophages (LMøs) proliferated poorly after PPD stimulation in both non‐vaccinated and BCG‐vaccinated animals while robust proliferation to PPD was observed when T cells were co‐cultured with peritoneal macrophages (PMøs). Macrophages within the lung compartment appear to regulate the response of T cells irrespective of the vaccination status in guinea pigs. Taken together, our results suggest that type I cytokine mRNA expression is not associated with vaccine‐induced protection in the low‐dose guinea pig model of tuberculosis.

Autophagy: a new frontier in research in lung diseases.

Pseudomonas aeruginosa infection in cystic fibrosis: diagnostic and prognostic significance of Pseudomonas aeruginosa precipitins determined by means of crossed immunoelectrophoresis. Scand J Respir Dis 1977;58:65–79. 4. Fomsgaard A, Hoiby N, Shand GH, Conrad RS, Galanos C. Longitudinal study of antibody response to lipopolysaccharides during chronic Pseudomonas aeruginosa lung infection in cystic fibrosis. Infect Immun 1988;56:2270–2278. 5. Frederiksen B, Koch C, Hoiby N. Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis. Pediatr Pulmonol 1997;23:330–335. 6. Frederiksen B, Lanng S, Koch C, Hoiby N. Improved survival in the Danish center-treated cystic fibrosis patients: results of aggressive treatment. Pediatr Pulmonol 1996;21:153–158. 7. Hansen CR, Pressler T, Hoiby N. Early aggressive eradication therapy for intermittent Pseudomonas aeruginosa airway colonization in cystic fibrosis patients: 15 years experience. J Cyst Fibros 2008;7: 523–530. 8. Ren CL, Morgan WJ, Konstan MW, Schechter MS, Wagener JS, Fisher KA, Regelmann WE. Presence of methicillin resistant Staphylococcus aureus in respiratory cultures from cystic fibrosis patients is associated with lower lung function. Pediatr Pulmonol 2007;42:513– 518. 9. Dasenbrook EC, Merlo CA, Diener-West M, Lechtzin N, Boyle MP. Persistent methicillin-resistant Staphylococcus aureus and rate of FEV1 decline in cystic fibrosis. Am J Respir Crit Care Med 2008;178:814–821. 10. Sawicki GS, Rasouliyan L, Pasta DJ, Regelmann WE, Wagener JS, Waltz DA, Ren CL. The impact of incident methicillin resistant Staphylococcus aureus detection on pulmonary function in cystic fibrosis. Pediatr Pulmonol 2008;43:1117–1123. 11. Kappler M, Kraxner A, Reinhardt D, Ganster B, Griese M, Lang T. Diagnostic and prognostic value of serum antibodies against Pseudomonas aeruginosa in cystic fibrosis. Thorax 2006;61:684–688. 12. Proesmans M, Balinska-Miskiewicz W, Dupont L, Bossuyt X, Verhaegen J, Hoiby N, de Boeck K. Evaluating the ‘‘Leeds criteria’’ for Pseudomonas aeruginosa infection in a cystic fibrosis centre. Eur Respir J 2006; 27:937–943. 13. Pressler T, Karpati F, Granstrom M, Knudsen PK, Lindblad A, Hjelte L, Olesen HV, Meyer P, Høiby N. Diagnostic significance of measurements of specific IgG antibodies to Pseudomonas aeruginosa by three different serological methods. J Cyst Fibros 2009;8:37–42. 14. Ratjen F, Walter H, Haug M, Meisner C, Grasemann H, Doring G. Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis patients. Pediatr Pulmonol 2007; 42:249–255. 15. West SE, Zeng L, Lee BL, Kosorok MR, Laxova A, Rock MJ, Splaingard MJ, Farrell PM. Respiratory infections with Pseudomonas aeruginosa in children with cystic fibrosis: early detection by serology and assessment of risk factors. JAMA 2002;287:2958–2967. 16. Tramper-Stranders GA, van der Ent CK, Slieker MG, Slieker MG, Terheggen-Lagro SW, Teding van Berkhout F, Kimpen JL, Wolfs TF. Diagnostic value of serological tests against Pseudomonas aeruginosa in a large cystic fibrosis population. Thorax 2006;61: 689–693. 17. Douglas TA, Brennan S, Berry L, Winfield K, Wainwright CE, Grimwood K, Stick SM, Sly PD. Value of serology in predicting Pseudomonas aeruginosa infection in young children with cystic fibrosis. Thorax 2010;65:985–990.

A secondary wave of neutrophil infiltration causes necrosis and ulceration in lesions of experimental American cutaneous leishmaniasis

We evaluated the importance of neutrophils in the development of chronic lesions caused by L. Viannia spp. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R2 = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p<0.001 each). In contrast, reduced neutrophil infiltration via cyclophosphamide-mediated depletion led to more benign lesions and lower parasite loads compared to controls (p<0.001 each). Neutrophils of the second wave expressed significantly lower GM-CSF, reactive oxygen species and nitric oxide than those of the first wave, suggesting that they had less efficient anti-leishmania activity. However, there was increased inflammatory cytokines and expression of neutrophil proteases (myeloperoxidase, cathepsin G and elastase) in lesions during the second wave of neutrophil infiltration compared with the levels reached during the first wave (6h p.i.). This suggests that augmented neutrophil proteases and inflammatory cytokines during the secondary wave of neutrophils could contribute to skin inflammation, ulceration and necrosis in ACL. The overall results indicate that neutrophils were unable to clear the infection in this model, and that the second wave of neutrophils played an important role in the severity of ACL.