Variations in maternal adenylate cyclase genes are associated with congenital Zika syndrome in a cohort from Northeast, Brazil.

BACKGROUND: Vertical transmission of Zika virus (ZIKV) is associated with congenital malformations but the mechanism of pathogenesis remains unclear. Although host genetics appear to play a role, no genetic association study has yet been performed to evaluate this question. In order to investigate if maternal genetic variation is associated with Congenital Zika Syndrome (CZS), we conducted a case-control study in a cohort of Brazilian women infected with ZIKV during pregnancy. METHODS: A total of 100 women who reported symptoms of zika during pregnancy were enrolled and tested for ZIKV. Among 52 women positive for ZIKV infection, 28 were classified as cases and 24 as controls based on the presence or absence of CZS in their infants. Variations in the coding region of 205 candidate genes involved in cAMP signaling or immune response were assessed by high throughput sequencing and tested for association with development of CZS. RESULTS: From the 817 single nucleotide variations (SNVs) included in association analyses, 22 SNVs in 17 genes were associated with CZS under an additive model (alpha = 0.05). Variations c.319T>C (rs11676272) and c.1297G>A, located at ADCY3 and ADCY7 genes showed the most prominent effect. The association of ADCY3 and ADCY7 genes was confirmed using a Sequence Kernel Association Test to assess the joint effect of common and rare variations, and results were statistically significant after adjustment for multiple comparisons (P < 0.002). CONCLUSION: These results suggest that maternal ADCY genes contribute to ZIKV pathogenicity and influence the outcome of CZS, being promising candidates for further replication studies and functional analysis.

CD4 T cell subsets in the mucosa are CD28+Ki-67-HLA-DR-CD69+ but show differential infection based on alpha4beta7 receptor expression during acute SIV infection.

BACKGROUND: CD4 T cell depletion in the mucosa has been well documented during acute HIV and SIV infections. The demonstration the HIV/SIVcan use the alpha4beta7 receptor for viral entry suggests that these viruses selectively target CD4 T cells in the mucosa that express high levels of alpha4beta7 receptor. METHODS: Mucosal samples obtained from SIV infected rhesus macaques during the early phase of infection were used for immunophenotypic analysis. CD4 T cell subsets were sorted based on the expression of beta7 and CD95 to quantify the level of SIV infection in different subsets of CD4 T cells. Changes in IL-17, IL-21, IL-23 and TGFbeta mRNA expression was determined using Taqman PCR. RESULTS: CD4 T cells in the mucosa were found to harbor two major population of cells; -25% of CD4 T cells expressed the alpha4(+)beta7(hi) phenotype, whereas the rest of the 75% expressed an alpha4(+)beta7(int) phenotype. Both the subsets were predominantly CD28(+)Ki-67(-)HLA-DR(-) but CD69(+), and expressed detectable levels of CCR5 on their surface. Interestingly, however, alpha4(+)beta7(hi)CD4 T cells were found to harbor more SIV than the alpha4(+)beta7(int) subsets at day 10 pi. Early infection was associated with a dramatic increase in the expression of IL-17, and IL-17 promoting cytokines IL-21, IL-23, and TGFbeta that stayed high even after the loss of mucosal CD4 T cells. CONCLUSIONS: Our results suggest that the differential expression of the alpha4beta7 receptor contributes to the differences in the extent of infection in CD4 T cell subsets in the mucosa. Early infection is associated dysregulation of the IL-17 network in mucosal tissues involves other non-Th-17 cells that likely contributes to the pro-inflammatory environment in the mucosa during acute stages of SIV infection.

Anti-retroviral therapy fails to restore the severe Th-17: Tc-17 imbalance observed in peripheral blood during simian immunodeficiency virus infection.

BACKGROUND: Human immuno deficiency virus and simian immunodeficiency virus infections are characterized by a severe loss of Th-17 cells (IL-17(+)CD4(+) T cells) that has been associated with disease progression and systemic dissemination of bacterial infections. Anti-retroviral therapy (ART) has led to repopulation of CD4(+) T cells in peripheral tissues with little sustainable repopulation in mucosal tissues. Given the central importance of Th-17 cells in mucosal homeostasis, it is not known if the failure of ART to permanently repopulate mucosal tissues is associated with a failure to restore Th-17 cells that are lost during infection. METHODS: Dynamics of alpha4(+)beta7(hi) CD4(+) T cells in peripheral blood of SIV infected rhesus macaques were evaluated and compared to animals that were treated with ART. The frequency of Th-17 and Tc-17 cells was determined following infection and after therapy. Relative expression of IL-21, IL-23, and TGFbeta was determined using Taqman PCR. RESULTS: Treatment of SIV infected rhesus macaques with anti-retroviral therapy was associated with a substantial repopulation of mucosal homing alpha4(+)beta7(hi)CD4(+) T cells in peripheral blood. This repopulation, however, was not accompanied by a restoration of Th-17 responses. Interestingly, SIV infection was associated with an increase in Tc-17 responses (IL-17(+)CD8(+) T cells) suggesting to a skewing in the ratio of Th-17: Tc-17 cells from a predominantly Th-17 phenotype to a predominantly Tc-17 phenotype. Surprisingly, Tc-17 responses remained high during the course of therapy suggesting that ART failed to correct the imbalance in Th-17 : Tc-17 responses induced following SIV infection. CONCLUSIONS: ART was associated with substantial repopulation of alpha4(+)beta7(hi) CD4(+) T cells in peripheral blood with little or no rebound of Th-17 cells. On the other hand, repopulation of alpha4(+)beta7(hi) CD4(+) T cells was accompanied by persistence of high levels of Tc-17 cells in peripheral blood. The dysregulation of Th-17 and Tc-17 responses likely plays a role in disease progression.

Alpha4(+)beta7(hi)CD4(+) memory T cells harbor most Th-17 cells and are preferentially infected during acute SIV infection.

Human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) infections are believed to infect minimally activated CD4(+) T cells after viral entry. Not much is known about why SIV selectively targets these cells. Here we show that CD4(+) T cells that express high levels of the alpha4beta7 heterodimer are preferentially infected very early during the course of SIV infection. At days 2-4 post infection, alpha4(+)beta7(hi)CD4(+) T cells had approximately 5x more SIV-gag DNA than beta7(-)CD4(+) T cells. alpha4(+)beta7(hi)CD4(+) T cells displayed a predominantly central memory (CD45RA(-)CD28(+)CCR7(+)) and a resting (CD25(-)CD69(-)HLA-DR(-)Ki-67(-)) phenotype. Although the expression of detectable CCR5 was variable on alpha4(+)beta7(hi) and beta7(-)CD4(+) T cells, both CCR5(+) and CCR5(-) subsets of alpha4(+)beta7(hi) and beta7(-)CD4(+) T cells were found to express sufficient levels of CCR5 mRNA, suggesting that both these subsets could be efficiently infected by SIV. In line with this, we found similar levels of SIV infection in beta7(-)CD4(+)CCR5(+) and beta7(-)CD4(+)CCR5(-) T cells. alpha4beta7(hi)CD4(+) T cells were found to harbor most T helper (Th)-17 cells that were significantly depleted during acute SIV infection. Taken together, our results show that resting memory alpha4(+)beta7(hi)CD4(+) T cells in the blood are preferentially infected and depleted during acute SIV infection, and the loss of these cells alters the balance between Th-17 and Th-1 responses, thereby contributing to disease pathogenesis.

An early expansion of CD8alphabeta T cells, but depletion of resident CD8alphaalpha T cells, occurs in the intestinal epithelium during primary simian immunodeficiency virus infection.

OBJECTIVES: To evaluate changes in the phenotypic heterogeneity and function of CD8 T cells in the intestinal epithelium during primary SIV infection. DESIGN: Previous studies have shown an increased prevalence of CD8 T cells in the intestinal epithelium in HIV and SIV infections. As intestinal CD8 T cells are a heterogeneous population we evaluated their phenotypic distribution (CD8alphabeta, CD8alphaalpha) and function [interferon (IFN)-gamma production] during primary SIV infection. METHODS: The phenotype and functional potential of CD8 intestinal intraepithelial lymphocytes (IEL) prior to and following SIV infection were determined using flow cytometry. RESULTS: IEL were found to harbor CD8alphabetaCD3, CD8alphaalphaCD3 and CD8alphaalpha+CD3- T-cell subsets. Most of the CD8CD4 double positive IEL expressed CD8alphaalpha homodimers. In primary SIV infection the frequency of CD8alphabetaCD3 T cells increased dramatically whereas the frequency of CD8alphaalpha T cells declined. A higher frequency of CD8alphabetaKi-67 IEL was observed following SIV infection suggesting that local cell proliferation might have contributed to an increased prevalence of CD8alphabeta IEL. In contrast, a severe depletion of CD8alphaalphaCD4 IEL occurred which contributed to the depletion of CD8alphaalpha IEL. The CD8alphabeta IEL were the major producers of IFN-gamma in the intestinal epithelium and the frequency of IFN-gamma-producing CD8alphabeta IEL was enhanced considerably in primary infection. CONCLUSIONS: CD8alphabeta IEL may be important in generating early antiviral responses at the intestinal epithelium. However, alterations in CD8 T-cell subsets and their function may reflect early immunopathogenic events in the intestinal mucosa.

A predominant Th1 type of immune response is induced early during acute Helicobacter pylori infection in rhesus macaques.

BACKGROUND & AIMS: The immune response of gastric T cells during acute Helicobacter pylori infection has not been previously characterized. The aim of this study was to delineate the phenotypic and functional responses of gastric T cells during acute H. pylori infection of rhesus macaques. METHODS: Four monkeys were experimentally infected with H. pylori. Gastric biopsy specimens and peripheral blood samples were obtained 1 and 12 weeks after inoculation. Samples from 3 animals uninfected with H. pylori served as controls. The immunophenotypic changes and functional potential of CD4(+) and CD8(+) T cells in gastric mucosa and peripheral blood to produce cytokines (interleukin [IL]-2, IL-4, IL-13, interferon [IFN]-gamma, MIP-1beta, and tumor necrosis factor [TNF]-alpha) were determined at a single cell level using flow cytometry. RESULTS: An increase in CD4(+) T cells occurred in the gastric mucosa during acute H. pylori infection as early as 1 week after infection. Acute infection was characterized by a predominantly T helper (Th)1 (IL-2 and IFN-gamma) and proinflammatory (TNF-alpha and MIP-1beta) type of cytokine response and the absence of a Th2 type of response. CONCLUSIONS: A predominant Th1 type response was induced early during acute H. pylori infection and may contribute to the development of gastric disease.

Activated memory CD4(+) T helper cells repopulate the intestine early following antiretroviral therapy of simian immunodeficiency virus-infected rhesus macaques but exhibit a decreased potential to produce interleukin-2.

Using the simian immunodeficiency virus (SIV)-infected rhesus macaque model, we performed a longitudinal study to determine the effect of antiretroviral therapy on the phenotype and functional potential of CD4(+) T cells repopulating intestinal mucosa in human immunodeficiency virus infection. Severe depletion of CD4(+) and CD4(+) CD8(+) T cells occurred in the intestinal mucosa during primary SIV infection. The majority of these cells were of activated memory phenotype. Phosphonate 9-[2-(phosphomethoxypropyl]adenine (PMPA) treatment led to a moderate suppression of intestinal viral loads and repopulation of intestinal mucosa by predominantly activated memory CD4(+) T-helper cells. This repopulation was independent of the level of viral suppression. Compared to preinfection values, the frequency of naive CD4(+) T cells increased following PMPA therapy, suggesting that new CD4(+) T cells were repopulating the intestinal mucosa. Repopulation by CD4(+) CD8(+) T cells was not observed in either jejunum or colon lamina propria. The majority of CD4(+) T cells repopulating the intestinal mucosa following PMPA therapy were CD29(hi) and CD11ahi. A subset of repopulating intestinal CD4(+) T cells expressed Ki-67 antigen, indicating that local proliferation may play a role in the repopulation process. Although the majority of repopulating CD4(+) T cells in the intestinal mucosa were functionally capable of providing B- and T-cell help, as evidenced by their expression of CD28, these CD4(+) T cells were found to have a reduced capacity to produce interleukin-2 (IL-2) compared to the potential of CD4(+) T cells prior to SIV infection. Persistent viral infection may play a role in suppressing the potential of repopulating CD4(+) T cells to produce IL-2. Hence, successful antiretroviral therapy should aim at complete suppression of viral loads in mucosal lymphoid tissues, such as intestinal mucosa.

Gastrointestinal epithelium is an early extrathymic site for increased prevalence of CD34(+) progenitor cells in contrast to the thymus during primary simian immunodeficiency virus infection.

The objective of this study was to determine the effects of primary simian immunodeficiency virus (SIV) infection on the prevalence and phenotype of progenitor cells present in the gastrointestinal epithelia of SIV-infected rhesus macaques, a primate model for human immunodeficiency virus pathogenesis. The gastrointestinal epithelium was residence to progenitor cells expressing CD34 antigen, a subset of which also coexpressed Thy-1 and c-kit receptors, suggesting that the CD34(+) population in the intestine comprised a subpopulation of primitive precursors. Following experimental SIVmac251 infection, an early increase in the proportions of CD34(+) Thy-1(+) and CD34(+) c-kit+ progenitor cells was observed in the gastrointestinal epithelium. In contrast, the proportion of CD34(+) cells in the thymus declined during primary SIV infection, which was characterized by a decrease in the frequency of CD34(+) Thy-1(+) progenitor cells. A severe depletion in the frequency of CD4-committed CD34(+) progenitors was observed in the gastrointestinal epithelium 2 weeks after SIV infection which persisted even 4 weeks after infection. A coincident increase in the frequency of CD8- committed CD34(+) progenitor cells was observed during primary SIV infection. These results indicate that in contrast to the primary lymphoid organs such as the thymus, the gastrointestinal epithelium may be an early extrathymic site for the increased prevalence of both primitive and committed CD34(+) progenitor cells. The gastrointestinal epithelium may potentially play an important role in maintaining T-cell homeostasis in the intestinal mucosa during primary SIV infection.

Intracellular cytokine expression in the CD4+ and CD8+ T cells from intestinal mucosa of simian immunodeficiency virus infected macaques.

Isolated intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) from jejunum of SIV infected animals were examined for alterations in basal cytokine expression by RT-PCR. Remarkable changes in IFNgamma and IL-10 RNA levels were observed in IEL and LPL in SIV infection while IL-4 and IL-2 RNA levels remained unaltered. In addition, the CD4+ and CD8+ LPL were examined for intracellular cytokine production following mitogenic activation by flowcytometry. Both CD4+ and CD8+ T lymphocytes in intestinal mucosa retained the potential to produce IFNgamma in response to mitogenic stimulation in vitro, without a remarkable change in IL-4 production. The dominant IFNgamma cytokine response could be one of the major contributing factors in SIV associated enteropathy.

Intestinal intraepithelial lymphocytes are primed for gamma interferon and MIP-1beta expression and display antiviral cytotoxic activity despite severe CD4(+) T-cell depletion in primary simian immunodeficiency virus infection.

Intraepithelial lymphocytes (IEL) are a critical effector component of the gut-associated lymphoid tissue (GALT) and play an important role in mucosal immunity as well as in the maintenance of the epithelial cell integrity and barrier function. The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of rhesus macaques would cause alterations in the immunophenotypic profiles of IEL and their mitogen-specific cytokine (gamma interferon [IFN-gamma] and MIP-1beta) responses (by flow cytometry) and virus-specific cytotoxic T-cell (CTL) activity (by the chromium release assay). Virally infected IEL were detected through the entire course of SIV infection by in situ hybridization. Severe depletion of CD4(+) single-positive and CD4(+)CD8(+) double-positive T cells occurred early in primary SIV infection, which was coincident with an increased prevalence of CD8(+) T cells. This was in contrast to a gradual depletion of CD4(+) T cells in peripheral blood. The CD8(+) IEL were the primary producers of IFN-gamma and MIP-1beta and were found to retain their potential to produce both IFN-gamma and MIP-1beta through the entire course of SIV infection. SIV-specific CTL activity was detected in primary IEL at 1, 2, and 4 weeks post-SIV infection. These results demonstrated that IEL may be involved in generating antiviral immune responses early in SIV infection and in suppressing viral infection thereafter. Alterations in homeostasis in epithelia due to severe CD4(+) T-cell depletion accompanied by changes in the cytokine and chemokine production by IEL may play a role in the enteropathogenesis of SIV infection.

Gastrointestinal T lymphocytes retain high potential for cytokine responses but have severe CD4(+) T-cell depletion at all stages of simian immunodeficiency virus infection compared to peripheral lymphocytes.

Gastrointestinal complications in human immunodeficiency virus (HIV) infection are indicative of impaired intestinal mucosal immune system. We used simian immunodeficiency virus (SIV)-infected rhesus macaques as an animal model for HIV to determine pathogenic effects of SIV on intestinal T lymphocytes. Intestinal CD4(+) T-cell depletion and the potential for cytokine responses were examined during SIV infection and compared with results for lymphocytes from lymph nodes and blood. Flow cytometric analysis demonstrated severe depletion of CD4(+)CD8(-) single-positive T cells and CD4(+)CD8(+) double-positive T cells in intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL) during primary SIV infection which persisted through the entire course of SIV infection. In contrast, CD4(+) T-cell depletion was gradual in peripheral lymph nodes and blood. Flow cytometric analysis of intracellular gamma interferon (IFN-gamma) and interleukin-4 (IL-4) production following short-term mitogenic activation revealed that LPL retained same or higher capacity for IFN-gamma production in all stages of SIV infection compared to uninfected controls, whereas peripheral blood mononuclear cells displayed a gradual decline. The CD8(+) T cells were the major producers of IFN-gamma. There was no detectable change in the frequency of IL-4-producing cells in both LPL and peripheral blood mononuclear cells. Thus, severe depletion of CD4(+) LPL and IEL in primary SIV infection accompanied by altered cytokine responses may reflect altered T-cell homeostasis in intestinal mucosa. This could be a mechanism of SIV-associated enteropathy and viral pathogenesis. Dynamic changes in intestinal T lymphocytes were not adequately represented in peripheral lymph nodes or blood.