Circulating inflammatory monocytes contribute to impaired influenza vaccine responses in HIV-infected participants

Abstract

Objective: Antibody responses are often impaired in old age and in HIV-positive (HIV+) infection despite virologic control with antiretroviral therapy but innate immunologic determinants are not well understood.

Design: Monocytes and natural killer cells were examined for relationships to age, HIV infection and influenza vaccine responses.

Methods: Virologically suppressed HIV+ (n = 139) and HIV-negative (HIV-) (n = 137) participants classified by age as young (18-39 years), middle-aged (40-59 years) and old (≥60 years) were evaluated preinfluenza and postinfluenza vaccination.

Results: Prevaccination frequencies of inflammatory monocytes were highest in old HIV+ and HIV-, with old HIV+ exhibiting higher frequency of integrin CD11b on inflammatory monocytes that was correlated with age, expression of C-C chemokine receptor-2 (CCR2) and plasma soluble tumor necrosis factor receptor-1 (sTNFR1), with inverse correlation with postvaccination influenza H1N1 antibody titers. Higher frequencies of CD11b+ inflammatory monocytes (CD11b(hi), >48.4%) compared with low frequencies of CD11b+ inflammatory monocytes (<15.8%) was associated with higher prevaccination frequencies of total and inflammatory monocytes and higher CCR2 MFI, higher plasma sTNFR1 and CXCL-10 with higher lipopolysaccharide stimulated expression of TNFα and IL-6, concomitant with lower postvaccination influenza antibody titers. In HIV+ CD11b(hi) expressers, the depletion of inflammatory monocytes from peripheral blood mononuclear cells resulted in enhanced antigen-specific CD4+ T-cell proliferation. Immature CD56(hi) natural killer cells were lower in young HIV+ compared with young HIV- participants.

Conclusion: Perturbations of innate immunity and inflammation signified by high CD11b on inflammatory monocytes are exacerbated with aging in HIV+ and negatively impact immune function involved in Ab response to influenza vaccination.

Fig. 1.. Frequencies of innate cell subsets in healthy controls and HIV+ participants at baseline.

Cryopreserved PBMC from HIV-infected and healthy participants (n=276, HIV+ n=139, HC, n=137) from different age groups (young; HIV+,Y+ n=28, HC, Y – n=40, middle aged; HIV+, M+ n=67, HC, M – n=56, old; HIV+, O+ n=44, HC, O – n=41) at baseline (pre vaccination) were thawed and rested overnight followed by ex-vivo staining with mAb. (a) Flow cytometric dot plots showing the gating strategy for monocytes from live CD45⁺ cells. Monocyte subsets are gated from live CD45⁺HLADR⁺CD3⁻CD56⁻ and based on CD14⁺ and CD16⁺, they are defined as Classical (CM, CD14hiCD16−), inflammatory (inflammatory monocytes, CD14⁺CD16⁺) and nonclassical (NCM, CD14loCD16hi). (b) Histogram depicting CD11b expression on classical monocytes (black solid fill), inflammatory monocytes (grey solid fill) and nonclassical (solid line). (d–f) Frequencies of monocyte subsets at T0 in HIV+ and HC participants. (G) natural killer cells were gated from Live CD45⁺CD3⁻CD8⁻CD14⁻cells. Based on CD56⁺ and CD16⁺, three populations namely, CD56^hi immature natural killer cells, CD56^dimCD16^neg and CD56^dim CD16⁺ mature natural killer cells were analyzed. (h) Frequencies of CD56^hiCD16⁻ natural killer cells in HIV+ and HC at baseline. Stars indicate the level of significance. *P<0.05, **P<0.01, ***P<0.001. HC, healthy controls.

Fig. 2.. Elevated frequencies of CD11b expression on monocytes in HIV+ participants at baseline.

Frequencies of (a) CD11b⁺ total monocytes and (b) CD11b⁺ inflammatory monocytes at baseline in all age groups in HIV+ and HC participants. Correlation between age and frequencies of (C) CD11b⁺ total monocytes and (d) CD11b⁺ inflammatory monocytes at baseline in HIV+ participants by Spearman’s correlation test. (e) Correlation between dual expression of CD11b and CCR2 on inflammatory monocytes in HIV+ participants at baseline. (f) Correlation between frequencies of CD11b⁺ inflammatory monocytes and plasma soluble TNFrI in HIV+ participants. Stars indicate the level of significance. *P<0.05, **P<0.01, ***P<0.001

Fig. 3.. HIVR participants expressing high CD11b on inflammatory monocytes have lower antibody responses post vaccination.

(a) Correlation between frequencies of CD11b⁺ inflammatory monocytes at baseline and hemagglutination inhibition (HAI) antibody titers at week 4 postvaccination in HIV+ participants by Spearman correlation test. (b) HIV+ participants were categorized as those with high frequencies of CD11b⁺ inflammatory monocytes (upper quartile 75%, frequency of CD11b⁺ inflammatory monocytes >48.4%, n=34) and those with low frequency of CD11b⁺ inflammatory monocytes (lower quartile 25%, CD11b⁺ inflammatory monocytes <15.8, n=33). (c) Comparison of H1N1 antibody titer at T0, T1 and T2 between CD11b^hi expressers and CD11b^lo expressers. (d) Frequencies of total monocytes and inflammatory monocytes in CD11b^hi expressers and CD11b^lo expressers at baseline. (e) CCR2 MFI expression on total monocytes, inflammatory monocytes and classical monocytes in CD11b^hi expressers and CD11b^lo expressers at baseline. (f) sTNFR1 levels in plasma of HIV+ CD11b^hi expressers and CD11b^lo expressers at baseline. (g) CXCL-10 gene expression in sorted inflammatory monocytes from CD11b^hi expressers (n=4) and CD11b^lo expressers (n=4) at baseline. Stars indicate the level of significance.

Fig. 4.. CD11b expression on inflammatory monocytes is associated with inflammatory profile in HIV infection at baseline and inhibit antigen-specific T-cell proliferation.

(a) PBMC from HIV+ CD11b^hi expressers (n=4) and CD11b^lo expressers (n=4) were thawed, rested and CD14⁺ CD16⁺ monocytes were sorted and stimulated with LPS (50 ng/ml) for 6 h for gene expression analysis and for 22 h for cytokine estimation in culture supernatants. (b) Expression levels of cytokine genes TNFα and IL6 by real time PCR. (c) Culture supernatants following stimulation with LPS were analyzed for TNFα and IL-6 using commercially ELISA kit. PBMC from CD11b^hi expressers (n=4) at T1 were thawed, rested and CD14⁺ CD16⁺ inflammatory monocytes were depleted by sorting. Inflammatory monocyte depleted and untouched PBMC was stained with cell trace dye and stimulated with H1N1 Ag (5 μg/ml) for 5 days and CD4⁺ T-cell proliferation assessed by Cell trace. (d) Representative histogram of whole PBMC and inflammatory monocyte-depleted PBMC from a single HIV+ CD11b^hi expresser showing CD4⁺ T-cell proliferation using Cell Trace violet. (e) Frequencies of Cell Trace^dim CD4⁺ T cells and (f) proliferation index in PBMC and inflammatory monocytes depleted PBMC of CD11b^hi expressers.