Resume

• 2004

  Doctor of Philosophy (Ph.D.)

  Immunology

  Pontificia Universidad Javeriana

• 1995

  Doctor of Medicine (M.D.)

  Pontificia Universidad Javeriana

• PCR

  Genetics

  Cell Culture

  Cell Biology

  Immunology

  Biochemistry

  Western Blotting

  Science

  Tissue Culture

  Scientific Writing

  Research

  Flow Cytometry

  Molecular Biology

  Statistics

  A role for mucosal IL-22 production and Th22 cells in HIV-associated mucosal immunopathogenesi

  Interleukin-22 (IL-22) is a cytokine with epithelial reparative and regenerative properties that is produced by Th22 cells and by other immune cell subsets. Therefore

  we explored the hypothesis that disruption of the gut barrier during HIV infection involves dysregulation of these cells in the gastrointestinal mucosa. Sigmoid IL-22-producing T cell and Th22 cells were dramatically depleted during chronic HIV infection

  epithelial integrity was compromised

  and microbial translocation was increased. These alterations were reversed after long-term antiretroviral therapy. While all mucosal IL-22-producing T-cell subsets were also depleted very early during HIV infection

  at these early stages IL-22 production by non-T-cell populations (including NKp44+ cells) was increased and gut epithelial integrity was maintained. Circulating Th22 cells expressed a higher level of the HIV co-receptor/binding molecules CCR5 and α4β7 than CD4+ T-cell subsets in HIV-uninfected participants

  but this was not the case after HIV infection. Finally

  recombinant IL-22 was protective against HIV and tumor necrosis factor-α-induced gut epithelial damage in a validated in vitro gut epithelial system. We conclude that reduced IL-22 production and Th22 depletion in the gut mucosa are important factors in HIV mucosal immunopathogenesis.

  A role for mucosal IL-22 production and Th22 cells in HIV-associated mucosal immunopathogenesi

  We have previously shown that human myeloid dendritic cells treated with purified rotavirus induce an allogenic Th1 response. To determine if rotavirus in the context of an intestinal microenvironment modulates the function of dendritic cells

  we treated these cells with supernatants from non-infected or infected Caco-2 cells and studied their capacity to promote Th1 or Th2 responses. Dendritic cells treated with supernatants from rotavirus-infected Caco-2 cells promoted a significantly lower Th1 response

  in comparison with those treated with purified rotavirus. We wanted to establish if TGF-β1

  induced

  or TSLP

  not induced

  during rotavirus infection

  could mediate this effect. Neutralization of TGF-β but not TSLP in the supernatant prior to treatment of dendritic cells increased their capacity to promote a Th1 response. The results suggest that the TGF-β1 induced by rotavirus could be an immune evasion mechanism

  and may partially explain the poor rotavirus-specific T cell response we have previously evidenced.

  Human Myeloid Dendritic cells treated with supernatants of Rotavirus infected Caco-2 cells induce a poor Th1 response

  In a double blind trial

  319 fully immunized children received two doses of either placebo or 10(6.7) focus-forming units of the attenuated RIX4414 human rotavirus (RV) vaccine (\"all-in-one\" formulation). Plasma RV-specific IgA (RV IgA)

  stool RV IgA

  and circulating total and RV memory B cells (CD19+ IgD+/- CD27+) with an intestinal homing phenotype (alpha4beta7+ CCR9+/-) were measured

  after the first and second doses

  as potential correlates of protection. After the first and/or second dose

  54% of vaccinees and 13% of placebo recipients had plasma RV IgA. Before vaccination

  most (95%) of the children (of both study groups) were breast-fed and had stool RV IgA (68.64%). Coproconversion (4-fold increase) after the first and/or second dose was observed in 32.7% of vaccinees and 17.4% of placebo recipients. No significant difference was seen when comparing the frequencies of any subset of memory B cells between vaccinees and placebo recipients. Statistically significant weak correlations were found between plasma RV IgA titers and coproconversion

  and several subsets of memory B cells. The vaccine provided 74.8% protection (95% confidence interval

  30.93-92.62) against any RV gastroenteritis and 100% protection (95% confidence interval

  14.53-100) against severe RV gastroenteritis. When vaccinees and placebo recipients were considered together

  a correlation was found between protection from disease and plasma RV IgA measured after dose 2 and RV memory (IgD- CD27+ alpha4beta7+ CCR9+) circulating B cells measured after dose 1. However

  the correlation coefficients for both tests were low (<0.2)

  suggesting that other factors are important in explaining protection from disease.

  Evaluation of circulating intestinally committed memory B cells in children vaccinated with an attenuated human rotavirus vaccine

  OBJECTIVE: To identify HLA-DRB1 alleles contributing to susceptibility to multiple sclerosis (MS) in a Colombian population and to estimate the common effect size of HLA class II on MS susceptibility in Latin American populations through a meta-analysis.\nMETHODS: A total of 65 Colombian patients with MS and 184 matched controls were included. HLA-DRB1 typing was done using the sequence-specific oligonucleotide probe method. A bivariate and a multivariate logistic regression analyses were done. Case-control studies performed in Latin America were searched up to January 2009 through a systematic review of the literature. Effect summary odds ratios (ORs) and 95% confidence intervals (CIs) were obtained by means of the random effect model.\nRESULTS: A total of 464 cases and 2581 controls from 7 studies and the results of the present study in Colombians were analyzed. HLA-DRB1*15 (OR: 2.3; 95% CI: 1.68-3.07; p<0.001) and HLA-DQB1*06 (OR: 2.2; 95% CI: 1.54-3.07; p<0.001) groups as well as DRB1*1501 (OR: 2.6; 95% CI: 1.67-4.02; p<0.001)

  DRB1*1503 (OR: 2.2; 95% CI: 1.39-3.62; p=0.001) and DQB1*0602 (OR: 2.5; 95% CI: 1.66-3.71; p<0.001) alleles were found to be risk factors for MS. The myelin basic protein immunodominant sequence (221)VHFFKNIVT(229) was predicted to strongly and simultaneously bind to HLA-DRB1*1501 and *1503.\nCONCLUSION: The current study highlights the effect size of HLA class II in MS in Latin America and confirms similar allelic risk factors across diverse populations. Receptor-ligand interactions in the HLA-antigenic peptide complex could have potential predictive and therapeutical implications.

  HLA class II polymorphism in Latin American patients with multiple sclerosis

  Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.

  •\tLTβRdeficiency in the myeloid compartment alters T cell responses during a viral challenge without affecting antigen-specific IgA responses

  Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC

  the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda(-/-) GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda(-/-) GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition

  we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation

  demonstrating the importance of apoptosis in GC homeostasis and clonal selection.

  AID and Caspase 8 shape the Germinal Center response through Apoptosis

  The mechanisms that contribute to the maintenance of serological memory are still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM(+) and CD27- subpopulations

  which are associated with autoimmune diseases pathogenesis. In patients with autoimmune diseases treated with Rituximab (RTX)

  some autoantibodies (auto-Abs) decrease after treatment

  but other auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus

  maintenance of autoimmune and pathogen-specific serological memory may depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific mBc and antigen-specific Abs of different isotypes have not been simultaneously assessed in patients after RTX treatment. To study the relationship between mBc subpopulations and serological memory we characterized total

  RV- and tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune diseases before and after treatment with RTX. We also measured total

  RV- and TT-Abs

  and some auto-Abs by kinetic nephelometry

  ELISA

  and EliA tests

  respectively. Minor differences were observed between the relative frequencies of RV-mBc in healthy controls and patients with autoimmune disease. After RTX treatment

  naïve Bc and total

  RV- and TT-specific mBc [IgM(+)

  switched (IgA(+)/IgG(+))

  IgM(+) only

  IgD(+) only

  and CD27- (IgA(+)/IgG(+)/IgM(+))] were significantly diminished. An important decrease in total plasma IgM and minor decreases in total IgG and IgA levels were also observed. IgM rheumatoid factor

  IgG anti-CCP

  and IgG anti-dsDNA were significantly diminished. In contrast

  RV-IgA

  RV-IgG and RV-IgG1

  and TT-IgG titers remained stable. In conclusion

  in patients with autoimmunity

  serological memory against RV and TT seem to be maintained by long-lived plasma cells

  unaffected by RTX

  and an important proportion of total IgM and serological memory against some auto-antigens seem to be maintained by short-lived plasma cells

  dependent on mBc precursors depleted by RTX.

  Simultaneous assessment of rotavirus-specific memory B cells and serological memory alter B cell depletion therapy with rituximab

  Gradients of the sphingolipid sphingosine-1-phosphate (S1P) are responsible for the egress of lymphocytes from lymph nodes by activating the S1P1 receptor expressed on the surface of lymphocytes. Small molecule drugs that downregulate S1P receptors induce the sequestration of lymphocytes within lymph nodes

  thus preventing lymphocytes from accessing sites of inflammation. In particular

  FTY720

  a pan-S1P receptor agonist

  has been efficacious in the treatment of multiple sclerosis as well as its animal model

  experimental autoimmune encephalomyelitis (EAE)

  by virtue of its ability to restrain lymphocytes within the lymph nodes

  thus precluding their migration into the CNS. However

  multiple leukocyte subsets express S1P receptors of varying types

  and although it is beneficial to prevent transmigration of proinflammatory lymphocytes into the CNS

  allowing access of regulatory leukocyte subsets to the CNS is desirable. In this study

  we show that an S1P1-specific agonist (AUY954) is clinically efficacious in ameliorating pre-established EAE in SJL/J mice. Efficacy of AUY954 correlated with a reduction of lymphocytes in the CNS

  but access of plasmacytoid dendritic cells (pDCs) to the CNS was unimpaired

  and the presence of pDCs was found to be an important cofactor in mediating the clinical efficacy of AUY954. These results indicate that pDCs are important in quieting autoimmune responses during EAE

  and that trafficking inhibitors that are permissive for pDC accumulation in the CNS may be of therapeutic value for the treatment of multiple sclerosis.

  A Sphingosine-1-phosphate receptor 1-directed agonist reduces central nervous system inflammation in a plasmacytoid dendritic cell-dependent manner

  We have previously studied B cells

  from people and mice

  that express rotavirus-specific surface immunoglobulin (RV-sIg) by flow cytometry with recombinant virus-like particles that contain green fluorescent protein. In the present study we characterized circulating B cells with RV-sIg in children with acute and convalescent infection. During acute infection

  circulating RV-sIgD(-) B cells are predominantly large

  CD38(high)

  CD27(high)

  CD138(+/-)

  CCR6(-)

  alpha4beta7(+)

  CCR9(+)

  CCR10(+)

  cutaneous lymphocyte antigen-negative (CLA(-))

  L-selectin(int/-)

  and sIgM(+)

  sIgG(-)

  sIgA(+/-) lymphocytes. This phenotype likely corresponds to gut-targeted plasma cells and plasmablasts. During convalescence the phenotype switches to small and large lymphocytes

  CD38(int/-)

  CD27(int/-)

  CCR6(+)

  alpha4beta7(+/-)

  CCR9(+/-) and CCR10(-)

  most likely representing RV-specific memory B cells with both gut and systemic trafficking profiles. Of note

  during acute RV infection both total and RV-specific murine IgM and IgA antibody-secreting cells migrate efficiently to CCL28 (the CCR10 ligand) and to a lesser extent to CCL25 (the CCR9 ligand). Our results show that CCR10 and CCR9 can be expressed on IgM as well as IgA antibody-secreting cells in response to acute intestinal infection

  likely helping target these cells to the gut. However

  these intestinal infection-induced plasmablasts lack the CLA homing receptor for skin

  consistent with mechanisms of differential CCR10 participation in skin T versus intestinal plasma cell homing. Interestingly

  RV memory cells generally lack CCR9 and CCR10 and instead express CCR6

  which may enable recruitment to diverse epithelial sites of inflammation.

  Maturation and Trafficking Markers on Rotavirus-Specific B cells during Acute Infection and Convalescence in Children

  The largest mucosal surface in the body is in the gastrointestinal tract

  a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues

  B cells respond to cytokines

  sometimes in the absence of T-cell help

  undergo class switch recombination of their immunoglobulin receptor to IgA

  and differentiate to become plasma cells. However

  IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA(+) plasma cells also produce the antimicrobial mediators tumour-necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS)

  and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA(+) plasma cells can be recapitulated in vitro in the presence of gut stroma

  and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-α and iNOS in B-lineage cells resulted in a reduction in IgA production

  altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut

  and extend the repertoire of protective responses exhibited by some B-lineage cells.

  Acquisition of a multifunctional TNFα/iNOS-producing IgA+ plasma cell phenotype in the gut

  We quantified circulating total

  rotavirus (RV) and Tetanus toxin (TT) memory B cells (mBc) in healthy adults using a limiting dilution assay (LDA) and a flow cytometry assay (FCA) that permit evaluation of both CD27+ and CD27- mBc. RV mBc were enriched in the CD27-

  IgG+ and in the CD27+

  IgM+ subsets. The numbers of RV mBc were higher by FCA than by LDA and results of the two assays did not correlate. TT IgGmBc and RV IgA mBc determined by FCA and by LDA correlated with TT plasma IgG and RV plasma IgA

  respectively. The mean ratio of specific mBc/mug/ml of the corresponding plasma immunoglobulin was lower for TT IgG than for RV IgA mBc. Our studies contribute to understand the relationship between circulating mBc and serological memory

  and enhance our capacity to develop better correlates of protection against RV disease.

  Characterization of RV specific B cells and their relation with serological memory

  Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC)

  and \"danger signals\" released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-β1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others)

  but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV

  and VP6 co-localized with CD63 in RV-infected cells

  suggesting that this viral protein is associated with the MV

  and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children

  suggesting that these MV are released by RV-infected cells in vivo. Moreover

  fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-β

  because they were reversed by treatment of the T cells with the TGF-β-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous

  with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18 g/mL)

  and denser vesicles (>1.24 g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity.

  Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T cell function

  Innate immune responses provoke the accumulation of leukocytes at sites of inflammation. In addition to monocytes and granulocytes

  B cells also participate in antimicrobial innate immune responses; however

  the mechanisms for accumulation of B cells to sites of inflammation are not well understood. To study B cell accumulation following systemic inflammation

  we used a model synthetic ligand that stimulates a specific pattern recognition molecule

  nucleotide-binding oligomerization domain-containing protein 1 (Nod1). Upon exposure to Nod1 agonists

  both B cells and neutrophils rapidly accumulate within the spleen

  and dendritic cells migrate into the periarterial lymphoid sheath. Nod1 stimulation led to a marked increase in several chemokines within the spleen

  including CXCL13

  CCL2

  and CCL20. Whereas the lymphotoxin pathway was critical for the induction of the B cell chemoattractant CXCL13 in response to Nod1 agonists

  B cell accumulation within the spleen following Nod1-induced systemic inflammation was independent of the lymphotoxin pathway. In contrast

  a CCR6/CCL20 chemokine loop instructed rapid increase of B cells in the spleen in response to systemic administration of Nod1 agonists in a TNF-α-dependent manner. Moreover

  CCR6 was required to regulate Nod1-mediated B cell responses. These results reveal a novel mechanism of B cells during inflammation and shed light on how B cells participate in innate immune responses to microbial stimulation.

  A TNFα-CCL20-CCR6 axis regulates NOD1-induced B cell responses

  BACKGROUND: The foreskin is the site of most HIV acquisition in uncircumcised heterosexual men. Although HIV-exposed

  seronegative (HESN) uncircumcised men demonstrate HIV-neutralizing IgA and increased antimicrobial peptides (AMPs) in the foreskin prepuce

  no prospective studies have examined the mucosal immune correlates of HIV acquisition.\nMETHODS: To assess the association of foreskin immune parameters with HIV acquisition

  antimicrobial peptides and IgA with the capacity to neutralize a primary clade C HIV strain were quantified by blinded investigators

  using sub-preputial swabs collected longitudinally during a randomized trial of male circumcision for HIV prevention in Rakai

  Uganda.\nRESULTS: Participants were 99 men who acquired HIV (cases) and 109 randomly selected controls who remained HIV seronegative. At enrollment

  44.4% of cases vs. 69.7% of controls demonstrated IgA neutralization (adjusted OR = 0.31; 95% CI

  0.16-0.61). IgA neutralization was detected in 38.7% of cases and 70.7% of controls at the last seronegative case visit prior to HIV acquisition and the comparable control visit (adjusted OR 0.21; 95% CI

  0.11-0.39). Levels of the α-defensins and secretory leukocyte protease inhibitor (SLPI) were over ten-fold higher in the foreskin prepuce of cases who acquired HIV

  both at enrollment (mean 4.43 vs. 3.03 and 5.98 vs. 4.61 log(n) pg/mL

  P = 0.005 and 0.009

  respectively)

  and at the last seronegative visit (mean 4.81 vs. 3.15 and 6.46 vs. 5.20 log(n) pg/mL

  P = 0.0002 and 0.013).\nCONCLUSIONS: This prospective

  blinded analysis is the first to assess the immune correlates of HIV acquisition in the foreskin. HIV-neutralizing IgA

  previously associated with the HESN phenotype

  was a biomarker of HIV protection

  but other HESN associations correlated with increased HIV acquisition. This emphasizes the importance of prospective epidemiological studies or in vitro tissue studies to define the impact of mucosal parameters on HIV risk.

  HIV Acquisition Is Associated with Increased Antimicrobial Peptides and Reduced HIV Neutralizing IgA in the Foreskin Prepuce of Uncircumcised Men.

  Fritz JH

  Gommerman JL

  Re-thinking the functions of IgA(+) plasma cells

  Rojas MD

  PhD

  University of Toronto

  Universidad del Rosario

  Trinity College in The University of Toronto

  Department of Immunology Faculty of Medicine

  Research Associate

  University of Toronto

  Deparment of Immunology Faculty of Medicine

  Assistant Professor

  Universidad del Rosario

  Department of Immunology Faculty of Medicine U of T

  Postdoctoral Fellow

  University of Toronto

  Toronto

  ON

  Contemporary Issues in Health Science Course

  Professor

  Trinity College in The University of Toronto

  Spanish

  English

  9th dsRNA virus simposyum. Travel Award

  Travel grant

  Canadian Institutes of Health Research (CIHR) Fellowship Award in the area of Immflamatory Bowel Disease

  Multiple Sclerosis Society of Canada (MSSOC) Fellowship Award