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Papers by David Colón

Supplementary Figure S5. Generation of myeloid cell-specific TLR4 knockout mice. (A) Representati... more Supplementary Figure S5. Generation of myeloid cell-specific TLR4 knockout mice. (A) Representative results of PCR-based genotyping: 1.The LysM-Cre heterozygous mice were detected by genomic PCR which gives raise two bands at 700-bp and 350-bp; 2. The homozygous TLR4-floxed mice were recognized by genomic PCR rendering a 285-bp band. To confirm the efficiency of LysM-Cre-mediated TLR4 loxP recombination in macrophages, BMDM from LysM-Cre +/and LysM-Cre +/-/TLR4 fl/fl mice were stimulated with LPS (100 ng/mL) or PCX (30 µM) and after 24h were measured the cytokines production in the supernatant: (B) TNF-α and (C) IL-6. All values are means ± SEM (n=3) *P<0.05

blocks the M2 macrophage polarization induced by IL-4. BMDM cells were stimulated with LPS (100 n... more blocks the M2 macrophage polarization induced by IL-4. BMDM cells were stimulated with LPS (100 ng/mL-M1-like), PCX (30µM) or IL-4 (10 ng/mL) alone or with IL-4 plus LPS (100ng/mL) or plus PCX (10, 30, 100 µM), unstimulated cells acted as a negative control (M0). (A) Flow plots correspond to the CD206 + on F4/80 + cells (representative data of three independent experiments). (B) Quantification of the results from A. (C) Histogram plots comparing CD206 expression by MFI on BMDM F480 + cells incubated with IL-4 (blue line) or IL-4 plus PCX (red line) (representative of three independent experiments). (D) Quantification of the results from C. In the supernatants after 48 of culture M2 and M1 markers were measured: (E) CCL22, (F) IGF-1, (G) urea, (H) TNF-α, (I) IL-6, and (J) nitrite, respectively. All values are means ± SEM (n=3), *P<0.05.

Institute. The rows/genes were normalized and expressed as log 2. The hierarchical clustering usi... more Institute. The rows/genes were normalized and expressed as log 2. The hierarchical clustering using Pearson correlation was performed. Nitrite accumulation In the supernatants from BMDM cell culture the nitrite, a stable metabolite of NO, was quantify by the Griess method (1). Briefly, the Griess reagent (0.5% sulfanilic acid, 0.002% N-1-naphtyl-ethylenediamine dihydrochloride, 14% glacial acetic acid) was added in supernatants of BMDM cell culture (v/v). Absorbance was measured at 550 nm, and the nitrite concentration was determined using sodium nitrite as a standard. Arginase assay Arginase activity was indirectly determined by measuring the metabolite urea, a product of arginine degradation. The urea concentration was measured from the supernatants of BMDM cultures. Quantification was conducted using the enzymatic colorimetric assay urea (CE LABTEST) followed the manufacturer's recommendation. The optical density of the individual samples was measured at 600 nm (Spectra Max-250, Molecular Devices), and values expressed in mg/dL.

European Journal of Pharmacology

Sepsis survival in adults is commonly followed by immunosuppression and increased susceptibility ... more Sepsis survival in adults is commonly followed by immunosuppression and increased susceptibility to secondary infections. However, the long-term immune consequences of pediatric sepsis are unknown. Here, we compared the frequency of Tregs, the activation of the IL-33/ILC2s axis in M2 macrophages, and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. In contrast to adults, infant mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistically, increased IL-33 levels, Tregs expansion, and activation of ILC2s and M2-macrophages were observed in post-septic adults but not infant mice. Impaired IL-33 production in post-septic infant mice was associated with increased DNA-methylation on lung epithelial cells. Notably, IL-33 treatment boosted the expansion of Tregs and induced immunosuppression in infant mice. Clinically, adults but not pediatric post-septic patients exhibited higher count...

Journal for ImmunoTherapy of Cancer, 2020

BackgroundPrevious data have reported that the growth of established tumors may be facilitated by... more BackgroundPrevious data have reported that the growth of established tumors may be facilitated by postsepsis disorder through changes in the microenvironment and immune dysfunction. However, the influence of postsepsis disorder in initial carcinogenesis remains elusive.MethodsIn the present work, the effect of postsepsis on inflammation-induced early carcinogenesis was evaluated in an experimental model of colitis-associated colorectal cancer (CAC). We also analyzed the frequency and role of intestinal T regulatory cells (Treg) in CAC carcinogenesis.ResultsThe colitis grade and the tumor development rate were evaluated postmortem or in vivo through serial colonoscopies. Sepsis-surviving mice (SSM) presented with a lower colonic DNA damage, polyp incidence, reduced tumor load, and milder colitis than their sham-operated counterparts. Ablating Treg led to restoration of the ability to develop colitis and tumor polyps in the SSM, in a similar fashion to that in the sham-operated mice. ...

Shock, 2018

Neutrophils and inflammatory monocytes control sepsis by migration to the site of infection via t... more Neutrophils and inflammatory monocytes control sepsis by migration to the site of infection via their chemokine receptors. CCR5 is a chemokine receptor that is not expressed on neutrophils and inflammatory monocytes under homeostatic conditions. However, it has been demonstrated that CCR5 can become expressed on these cells during different models of inflammation. In the present study, we investigated if CCR5 is also expressed on neutrophil and inflammatory monocytes during sepsis, exerting an important role in the migration of these cells to the infectious focus. Using cecal ligation and puncture model to induce polymicrobial sepsis, we demonstrated that the expression of CCR5 is induced on CD11b þ Ly6G À Ly6C high inflammatory monocytes, but not on neutrophils (CD11b þ Ly6G þ Ly6C À). Furthermore, CCR5 plays an important role for the migration of the inflammatory monocytes to infection focus during sepsis. CCR5-expressing inflammatory monocytes migrate from the bone marrow to the circulation and then into the site of infection, where they phagocytize and kill the bacteria. Consequently, CCR5 À/À mice showed increased systemic inflammatory response and mortality compared to wild-type mice. These data therefore demonstrate a hitherto unrecognized protective role of CCR5 in sepsis.

The Journal of Infectious Diseases, 2019

Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an... more Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.

Paclitaxel is an antineoplastic agent widely used to treat several solid tumor types. The primary... more Paclitaxel is an antineoplastic agent widely used to treat several solid tumor types. The primary mechanism of action of paclitaxel is based on microtubule stabilization inducing cell-cycle arrest. Here, we use several tumor models to show that paclitaxel not only induces tumor cell-cycle arrest, but also promotes antitumor immunity. In vitro, paclitaxel reprogrammed M2-polarized macrophages to the M1-like phenotype in a TLR4-dependent manner, similarly to LPS. Paclitaxel also modulated the tumor-associated macrophage (TAM) profile in mouse models of breast and melanoma tumors; gene expression analysis showed that paclitaxel altered the M2-like signature of TAMs toward an M1-like profile. In mice selectively lacking TLR4 on myeloid cells, for example, macrophages (LysM-Cre+/−/TLR4fl/fl), the antitumor effect of paclitaxel was attenuated. Gene expression analysis of tumor samples from patients with ovarian cancer before and after treatment with paclitaxel detected an enrichment of genes linked to the M1 macrophage activation profile (IFNγ-stimulated macrophages). These findings indicate that paclitaxel skews TAMs toward an immunocompetent profile via TLR4, which might contribute to the antitumor effect of paclitaxel and provide a rationale for new combination regimens comprising paclitaxel and immunotherapies as an anticancer treatment.Significance: This study provides new evidence that the antitumor effect of paclitaxel occurs in part via reactivation of the immune response against cancer, guiding tumor-associated macrophages toward the M1-like antitumor phenotype.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5891/F1.large.jpg. Cancer Res; 78(20); 5891–900. ©2018 AACR.See related commentary by Garassino et al., p. 5729

Pharmacological research, Mar 12, 2016

Sepsis is one of the main causes of mortality in hospitalized patients. Despite the recent techni... more Sepsis is one of the main causes of mortality in hospitalized patients. Despite the recent technical advances and the development of novel generation of antibiotics, severe sepsis remains a major clinical and scientific challenge in modern medicine. Unsuccessful efforts have been dedicated to the search of therapeutic options to treat the deleterious inflammatory components of sepsis. Recent findings on neuronal networks controlling immunity raised expectations for novel therapeutic strategies to promote the regulation of sterile inflammation, such as autoimmune diseases. Interesting studies have dissected the anatomical constituents of the so-called "cholinergic anti-inflammatory pathway", suggesting that electrical vagus nerve stimulation and pharmacological activation of beta-2 adrenergic and alpha-7 nicotinic receptors could be alternative strategies for improving inflammatory conditions. However, the literature on infectious diseases, such as sepsis, is still controve...

Journal of Natural Products, 2016

Vestitol is an isoflavonoid isolated from Brazilian red propolis with potential anti-inflammatory... more Vestitol is an isoflavonoid isolated from Brazilian red propolis with potential anti-inflammatory activity. This study investigated the mechanism of action of vestitol on the modulation of neutrophil migration in the inflammatory process. Pre-treatment with vestitol at 1, 3, or 10 mg/kg reduced LPS- or mBSA-induced neutrophil migration and the release of CXCL1/KC and CXCL2/MIP-2 in vivo. Likewise, pre-treatment with vestitol at 1, 3, or 10 μM reduced the levels of CXCL1/KC and CXCL2/MIP-2 in macrophage supernatants in vitro. Moreover, the administration of vestitol (10 mg/kg) reduced leukocyte rolling and adherence in the mesenteric microcirculation of mice. The pre-treatment with vestitol (10 mg/kg) in iNOS(-/-) mice did not block its activity concerning neutrophil migration. With regard to the activity of vestitol on neutrophils isolated from the bone marrow of mice, there was a reduction on the chemotaxis of CXCL2/MIP-2 or LTB4-induced neutrophils and on calcium influx after pre-treatment with the compound at 3 or 10 μM. There was no change in CXCR2 expression by neutrophils treated with vestitol at 10 μM. These findings demonstrate that vestitol is a promising novel anti-inflammatory agent.

Toxicon, 2014

Local tissue reactions provoked by Bothrops venoms are characterized by edema, hemorrhage, pain, ... more Local tissue reactions provoked by Bothrops venoms are characterized by edema, hemorrhage, pain, and inflammation; however, the mechanisms of tissue damage vary depending upon the species of snake. Here, we investigated the mechanisms involved in the local inflammatory response induced by the Bothrops jararacussu venom (BjcuV). Female Swiss mice were injected with either saline, BjcuV (0.125-8 µg/paw) or loratadine (an H 1 receptor antagonist), compound 48/80 (for mast cell depletion), capsaicin (for C-fiber desensitization), infliximab (an anti-TNF-α antibody), indomethacin (a non-specific COX inhibitor), celecoxib (a selective COX-2 inhibitor) or fucoidan (a P-and L-selectins modulator) given before BjcuV injection. Paw edema was measured by plethysmography. In addition, paw tissues were collected for the measurement of myeloperoxidase activity, TNF-α and IL-1 levels, and COX-2 immunoexpression. The direct chemotactic effect of BjcuV and the in vitro calcium dynamic in neutrophils were also investigated. BjcuV caused an edematogenic response with increased local production of TNF-α and IL-1β as well as COX-2 expression. Both edema and neutrophil migration were prevented by pretreatment with indomethacin, celecoxib or fucoidan. Furthermore, BjcuV induced a direct in vitro neutrophil chemotaxis by increasing intracellular calcium. Therefore, BjcuV induces an early onset edema dependent upon prostanoid production and neutrophil migration.

Characterization of mature/immature macrophages subsets in the tumor microenvironment

Frontiers in Immunology, 2020

The Chikungunya virus (CHIKV) is a re-emerging arbovirus, in which its infection causes a febrile... more The Chikungunya virus (CHIKV) is a re-emerging arbovirus, in which its infection causes a febrile illness also commonly associated with severe joint pain and myalgia. Although the immune response to CHIKV has been studied, a better understanding of the virus-host interaction mechanisms may lead to more effective therapeutic interventions. In this context, neutrophil extracellular traps (NETs) have been described as a key mediator involved in the control of many pathogens, including several bacteria and viruses, but no reports of this important protective mechanism were documented during CHIKV infection. Here we demonstrate that the experimental infection of mouse-isolated neutrophils with CHIKV resulted in NETosis (NETs release) through a mechanism dependent on TLR7 activation and reactive oxygen species generation. In vitro, mouse-isolated neutrophils stimulated with phorbol 12-myristate 13-acetate release NETs that once incubated with CHIKV, resulting in further virus capture and neutralization. In vivo, NETs inhibition by the treatment of the mice with DNase resulted in the enhanced susceptibility of IFNAR −/− mice to CHIKV experimental acute infection. Lastly, by accessing the levels of MPO-DNA complex on the acutely CHIKV-infected patients, we found a correlation between the levels of NETs and the viral load in the blood, suggesting that NETs are also released in natural human infection cases. Altogether our findings characterize NETosis as a contributing natural process to control CHIKV acute infection, presenting an antiviral effect that helps to control systemic virus levels.

Background and Purpose: Sepsis survival in adults is commonly followed by immunosuppression and i... more Background and Purpose: Sepsis survival in adults is commonly followed by immunosuppression and increased susceptibility to secondary infections. Nonetheless, the long-term immune consequences of pediatric sepsis are unknown. We sought to investigate the role of age in the genesis of immunosuppression following sepsis. Experimental Approach: Here, we compared the frequency of Tregs, the activation of the IL-33/ILC2s axis in M2 macrophages, and the DNA methylation of epithelial lung cells from post-septic infant and adult mice. Likewise, sepsis-surviving mice were inoculated intranasally with Pseudomonas aeruginosa or by subcutaneous inoculation of the B16 melanoma cell line. Finally, blood samples from sepsis-surviving patients were collected and the concentrations of IL-33 and Tregs frequency were assessed. Key Results: In contrast to 6-week-old, 2-week-old mice were resistant to secondary infection and did not show impairment in tumour controls upon melanoma challenge. Mechanistic...

Molecular Biology Reports

BACKGROUND Neutrophil extracellular traps (NETs) are a recently discovered neutrophil defense mec... more BACKGROUND Neutrophil extracellular traps (NETs) are a recently discovered neutrophil defense mechanism which modulates several inflammatory conditions contributing to metabolic profile alterations. Therefore, the present study aimed to evaluate the production of NETs in obese patients and mice, verifying the possible mechanisms associated with the release of NETs by the adipose tissue. METHODS AND RESULTS The present study investigated NETs production in human adipose tissue and also showing the neutrophils using intravital microscopy in mouse epididymal adipose tissue. Blood and white adipose tissues were obtained from eutrophic and obese individuals and from mice. Lipid, glycemic and leukocyte profiles were evaluated, as well as the levels of NETs and its markers. Bioinformatics and proteomics analyses were performed and the identified key proteins were measured. The main findings showed that the inflammatory markers interleukin-8 (IL-8), heat shock protein 90 (HSP90) and the E1 heat shock protein family (HSPE1) can be modulated by the NETs levels in obesity. Obesity has also been associated with increased cholesterol, glucose intolerance, ionic calcium and NETs. We also observed an increase in catalase and a decreased superoxide dismutase activity. Bioinformatics and proteomics analyses revealed that IL-8, HSP90 and HSPE1 were associated with obesity, inflammation and NETs release. CONCLUSIONS In conclusion, the present study shows an increase in NETs production during obesity associated with important inflammatory markers in adipose.