PD-1 is a marker that has been shown to be associated with both IA and IE. We investigated PD-1 expression only in a subset of 18 patients. PD-1 expression levels on CD4 and CD8 T cells were higher in patients at baseline compared with controls and increased further at the 12-month follow-up visit (Figs. 5A, B). At study entry, frequency of PD-1+ CD4 T cells correlated with LPS levels (Fig. 5C), 16S rDNA (Fig. 5D), and activated CD4 T cells (Fig. 5E). Frequency of PD1+ CD8 T cells correlated with LPS (Fig. 5F) and frequency of activated CD8 T cells (Fig. 5G).
To gain insight into disease progression in the absence of ART, we determined the MT and immunologic markers in the 47 HIV+ children who completed 12 months of follow-up. CD4% and absolute counts remained unchanged as did plasma levels of LPS, bacterial DNA, and sCD14 (see Figure S1 A-D, Supplemental Digital Content, http://links.lww.com/QAI/A500). CD4 T-cell and monocyte IA also remained unchanged at 12 months after entry (see Figure S1 E-F, Supplemental Digital Content, http://links.lww.com/QAI/A500). There was a small but significant increase in CD8 T-cell activation (Fig. 2F) and IE (Fig. 5B) as described above, indicating that the immunologic profile was not completely stable. Clinically, this subgroup of children did not experience serious infections in this time span.
There is evidence in adults that failure of immune reconstitution after ART can be associated with persistent MT and ongoing IA.9 Our study in children shows that in the absence of ART, the more severely immunocompromised children in the IC3 group had the highest MT, with elevated levels of both plasma LPS and 16S rDNA, whereas in patients in IC1 group, measures of MT were comparable with healthy controls. This is the first report of 16S rDNA in HIV-infected, treatment-naive pediatric population in a resource-limited setting. Earlier, we also reported that plasma 16S rDNA was elevated compared with healthy volunteers in a treatment-experienced HIV+ pediatric US cohort, but the values were far lower than those observed here.24 A recent study in ART-treated, HIV-infected children in the United Kingdom34 also found low levels of 16S rDNA in low frequency, and gut-associated bacterial species were identified by sequencing the bacterial DNA. In broad range quantitative 16S rDNA PCR assays, there is a possibility of DNA contamination, exogenous or endogenous, for which only cloning or sequencing can confirm gut bacteria. In the present study, the correlation of the 16S rDNA with LPS and its absence in healthy children and in experimental negative controls all point to the observed 16S rDNA as being a true indicator of MT. Future studies that determine the bacterial species in the 16S rDNA in different regional settings are warranted.
Although many factors have been suggested to contribute to IA during chronic HIV/SIV infection, MT is considered to be a major cause for it.6,41−43 The microbial products can stimulate immune cells directly via pattern recognition receptors, such as toll-like receptors. As activated T cells have a relatively short half‐life, the persistent IA in aviremic long-term ART-treated patients has been attributed to ongoing antigenic stimulation from MT.6 MT is also associated with IA during the chronic phase of SIV infection in rhesus macaques,44 whereas chronic SIV infection of sooty mangabeys does not cause damage to the intestinal barrier or result in MT and does not cause IA. The relationship of gut MT with IA and disease progression, however, remains controversial. In our study, MT was correlated with generalized IA as evidenced by HLA-DR+ CD38+ expressing T cells, circulating levels of sCD14 (a marker of monocyte activation), and increased IE (PD-1). The correlation between MT and activated T-cell phenotype points toward polyclonal T-cell activation, contributed by MT either directly or indirectly via cytokines and chemokines. The levels of neither of the MT products (LPS and 16S rDNA) showed a change during the 12-month follow-up in the absence of ART. CD4 IA also remained unchanged, but CD8 IA increased along with the frequency of PD-1+ T cells. These data affirm that MT is evident even in immunologically stable children although it is worse in association with CD4 immune deficiency, suggesting a potentially deleterious effect of ongoing MT on disease progression. MT and IA have been associated with disease progression even in long-term nonprogresssor adults.45
In the present study, we found significantly higher expression of the exhaustion marker PD-1 in T cells of HIV-infected children compared with age-matched controls, and its expression was directly related with IA. IE is a component of aberrant IA in chronic HIV-1 infection, which is associated with ongoing viral replication. We and others have reported previously that PD-1 and IA are closely linked in HIV-infected adults.18,50 Further supporting the link between IA and IE is the fact that PD-1 is expressed by activated T cells, but not naive T cells.51 PD-1 expression was further increased at the 12-month follow-up. As PD-1 expression was also directly associated with both LPS and 16S rDNA, these data suggest that MT not only is associated with IA but also contributes to IE.
The authors thank Dr Daniel Douek at the Vaccine Research Center at National Institutes of Health who provided the plasmid DNA containing known copy numbers and sequence of the primers and probe for the PCR. They also thank the HIV− and the HIV-1+ children who participated in the study, their families, and their providers.
1. Kotler DP, Reka S, Clayton F. Intestinal mucosal inflammation associated with human immunodeficiency virus infection. Dig Dis Sci. 1993;38:1119–1127.
2. Heise C, Miller CJ, Lackner A, et al.. Primary acute simian immunodeficiency virus infection of intestinal lymphoid tissue is associated with gastrointestinal dysfunction. J Infect Dis. 1994;169:1116–1120.
3. Jiang W, Lederman MM, Hunt P, et al.. Plasma levels of bacterial DNA correlate with immune activation and the magnitude of immune restoration in persons with antiretroviral-treated HIV infection. J Infect Dis. 2009;199:1177–1185.
4. Ancuta P, Kamat A, Kunstman KJ, et al.. Microbial translocation
is associated with increased monocyte activation and dementia in AIDS patients. PLoS One. 2008;3:e2516.
5. Brenchley JM, Price DA, Schacker TW, et al.. Microbial translocation
is a cause of systemic immune activation in chronic HIV infection. Nat Med. 2006;12:1365–1371.
6. Cassol E, Malfeld S, Mahasha P, et al.. Persistent microbial translocation
and immune activation in HIV-1-infected South Africans receiving combination antiretroviral therapy. J Infect Dis. 2010;202:723–733.
7. Giorgi JV, Hultin LE, McKeating JA, et al.. Shorter survival in advanced human immunodeficiency virus type 1 infection is more closely associated with T lymphocyte activation than with plasma virus burden or virus chemokine coreceptor usage. J Infect Dis. 1999;179:859–870.
8. Fahey JL, Taylor JM, Manna B, et al.. Prognostic significance of plasma markers of immune activation, HIV viral load and CD4 T-cell measurements. AIDS. 1998;12:1581–1590.
9. Marchetti G, Bellistri GM, Borghi E, et al.. Microbial translocation
is associated with sustained failure in CD4+ T-cell reconstitution in HIV-infected patients on long-term highly active antiretroviral therapy. AIDS. 2008;22:2035–2038.
10. Rajasuriar R, Booth D, Solomon A, et al.. Biological determinants of immune reconstitution in HIV-infected patients receiving antiretroviral therapy: the role of interleukin 7 and interleukin 7 receptor alpha and microbial translocation
. J Infect Dis. 2010;202:1254–1264.
11. Mavigner M, Cazabat M, Dubois M, et al.. Altered CD4+ T cell homing to the gut impairs mucosal immune reconstitution in treated HIV-infected individuals. J Clin Invest. 2012;122:62–69.
12. Wallet MA, Rodriguez CA, Yin L, et al.. Microbial translocation
induces persistent macrophage activation unrelated to HIV-1 levels or T-cell activation following therapy. AIDS. 2010;24:1281–1290.
13. Papasavvas E, Pistilli M, Reynolds G, et al.. Delayed loss of control of plasma lipopolysaccharide levels after therapy interruption in chronically HIV-1-infected patients. AIDS. 2009;23:369–375.
14. Kramski M, Gaeguta AJ, Lichtfuss GF, et al.. Novel sensitive real-time PCR for quantification of bacterial 16S rRNA genes in plasma of HIV-infected patients as a marker for microbial translocation
. J Clin Microbiol. 2011;49:3691–3693.
15. Kestens L, Vanham G, Gigase P, et al.. Expression of activation antigens, HLA-DR and CD38, on CD8 lymphocytes during HIV-1 infection. AIDS. 1992;6:793–797.
16. Levacher M, Hulstaert F, Tallet S, et al.. The significance of activation markers on CD8 lymphocytes in human immunodeficiency syndrome: staging and prognostic value. Clin Exp Immunol. 1992;90:376–382.
17. Day CL, Kaufmann DE, Kiepiela P, et al.. PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature. 2006;443:350–354.
18. Sachdeva M, Fischl MA, Pahwa R, et al.. Immune exhaustion occurs concomitantly with immune activation and decrease in regulatory T cells in viremic chronically HIV-1-infected patients. J Acquir Immune Defic Syndr. 2010;54:447–454.
19. Anderson KV. Toll signaling pathways in the innate immune response. Curr Opin Immunol. 2000;12:13–19.
20. Marchetti G, Tincati C, Silvestri G. Microbial translocation
in the pathogenesis of HIV infection and AIDS. Clin Microbiol Rev. 2013;26:2–18.
21. Kourtis AP, Ibegbu CC, Wiener J, et al.. Role of intestinal mucosal integrity in HIV Transmission to infants through Breast-feeding: the BAN study. J Infect Dis 2013;208:653–661.
22. Sherman MP. New concepts of microbial translocation
in the neonatal intestine: mechanisms and prevention. Clin Perinatol. 2010;37:565–579.
23. Papasavvas E, Azzoni L, Foulkes A, et al.. Increased microbial translocation
in ≤180 days old perinatally human immunodeficiency virus-positive infants as compared with human immunodeficiency virus-exposed uninfected infants of similar age. Pediatr Infect Dis J. 30:877–882.
24. Pilakka-Kanthikeel S, Huang S, Fenton T, et al.. Increased gut microbial translocation
in HIV-infected children
persists in virologic responders and virologic failures after antiretroviral therapy. Pediatr Infect Dis J. 2012;31:583–591.
25. Anselmi A, Vendrame D, Rampon O, et al.. Immune reconstitution in human immunodeficiency virus type 1-infected children with different virological responses to anti-retroviral therapy. Clin Exp Immunol. 2007;150:442–450.
26. Baroncelli S, Galluzzo CM, Pirillo MF, et al.. Microbial translocation
is associated with residual viral replication in HAART-treated HIV+ subjects with <50copies/ml HIV-1 RNA. J Clin Virol. 2009;46:367–370.
27. CDC. WHO case definitions of HIV for surveillance and revised clinical staging and immunological classification of HIV-related disease in adults and children. Geneva, Switzerland: World Health Organization; 2007.
28. Selvaraj A, Kanthikeel SP, Pk B, et al.. Defective dendritic cell response to toll like receptor 7/8 agonists in perinatally HIV infected children. Pathog Dis. 2013;69:184–193.
29. Luzuriaga K, Sullivan JL. Pediatric HIV-1 infection: advances and remaining challenges. AIDS Rev. 2002;4:21–26.
30. de Martino M, Tovo PA, Galli L, et al.. Prognostic significance of immunologic changes in 675 infants perinatally exposed to human immunodeficiency virus. The Italian Register for Human Immunodeficiency Virus Infection in Children. J Pediatr. 1991;119:702–709.
31. Hainline C, Taliep R, Sorour G, et al.. Early Antiretroviral Therapy reduces the incidence of otorrhea in a randomized study of early and deferred antiretroviral therapy: evidence from the Children with HIV Early Antiretroviral Therapy (CHER) Study. BMC Res Notes. 2011;26:448.
32. Laughton B, Cornell M, Grove D, et al.. Early antiretroviral therapy improves neurodevelopmental outcomes in infants. AIDS. 2010;26:1685–1690.
33. Leinert C, Stahl-Hennig C, Ecker A, et al.. Microbial translocation
in simian immunodeficiency virus (SIV)-infected rhesus monkeys (Macaca mulatta). J Med Primatol. 39:243–251.
34. Fitzgerald F, Harris K, Doyle R, et al.. Short communication: Evidence that microbial translocation
occurs in HIV-infected children
in the United Kingdom. AIDS Res Hum Retroviruses. 2013;29:1589–1593.
35. Marchetti G, Cozzi-Lepri A, Merlini E, et al.. Microbial translocation
predicts disease progression of HIV-infected antiretroviral-naive patients with high CD4+ cell count. AIDS. 2011;25:1385–1394.
36. Lopez M, Soriano V, Peris-Pertusa A, et al.. Elite controllers display higher activation on central memory CD8 T cells than HIV patients successfully on HAART. AIDS Res Hum Retroviruses. 2011;27:157–165.
37. Hunt PW, Brenchley J, Sinclair E, et al.. Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy. J Infect Dis. 2008;197:126–133.
38. Guadalupe M, Reay E, Sankaran S, et al.. Severe CD4+ T-cell depletion in gut lymphoid tissue during primary human immunodeficiency virus type 1 infection and substantial delay in restoration following highly active antiretroviral therapy. J Virol. 2003;77:11708–11717.
39. Poles MA, Boscardin WJ, Elliott J, et al.. Lack of decay of HIV-1 in gut-associated lymphoid tissue reservoirs in maximally suppressed individuals. J Acquir Immune Defic Syndr. 2006;43:65–68.
40. Brenchley JM, Schacker TW, Ruff LE, et al.. CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. J Exp Med. 2004;200:749–759.
41. Brenchley JM, Douek DC. The mucosal barrier and immune activation in HIV pathogenesis. Curr Opin HIV AIDS. 2008;3:356–361.
42. Balagopal A, Philp FH, Astemborski J, et al.. Human immunodeficiency virus-related microbial translocation
and progression of hepatitis C. Gastroenterology. 2008;135:226–233.
43. Appay V, Sauce D. Immune activation and inflammation in HIV-1 infection: causes and consequences. J Pathol. 2008;214:231–241.
44. Estes JD, Harris LD, Klatt NR, et al.. Damaged intestinal epithelial integrity linked to microbial translocation
in pathogenic simian immunodeficiency virus infections. PLoS Pathog. 2010;6:e1001052.
45. Salgado M, Rallon NI, Rodes B, et al.. Long-term non-progressors display a greater number of Th17 cells than HIV-infected typical progressors. Clin Immunol. 2011;139:110–114.
46. Vesterbacka J, Nowak P, Barqasho B, et al.. Kinetics of microbial translocation
markers in patients on efavirenz or lopinavir/r based antiretroviral therapy. PLoS One. 2013;8:e55038.
47. Redd AD, Dabitao D, Bream JH, et al.. Microbial translocation
, the innate cytokine response, and HIV-1 disease progression in Africa. Proc Natl Acad Sci U S A. 2009;106:6718–6723.
48. Anas A, van der Poll T, de Vos AF. Role of CD14 in lung inflammation and infection. Crit Care. 2010;14:209.
49. Ayaslioglu E, Kalpaklioglu F, Kavut AB, et al.. The role of CD14 gene promoter polymorphism in tuberculosis susceptibility. J Microbiol Immunol Infect. 2013;46:–.
50. Sauce D, Almeida JR, Larsen M, et al.. PD-1 expression on human CD8 T cells depends on both state of differentiation and activation status. AIDS. 2007;21:2005–2013.
51. Agata Y, Kawasaki A, Nishimura H, et al.. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol. 1996;8:765–772.