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Impaired T cell responsiveness to interleukin-6 in hematological patients with invasive aspergillosis.

Camargo JF, Bhimji A, Kumar D, Kaul R, Pavan R, Schuh A, Seftel M, Lipton JH, Gupta V, Humar A, Husain S - PLoS ONE (2015)

Bottom Line: While IFN-γ/STAT1 signaling was similar between groups, naïve T cells from patients with IA, but not those with mucormycosis, exhibited reduced responsiveness to IL-6 as measured by STAT3 phosphorylation.Furthermore, IL-6 increased Aspergillus-induced IL-17 production in culture supernatants from healthy and hematological controls but not in patients with IA.Altogether, these observations suggest an important role for dectin-1 and the IL-6/STAT3 pathway in protective immunity against Aspergillus.

View Article: PubMed Central - PubMed

Affiliation: Transplant Infectious Diseases, Multi-Organ Transplant Program, University Health Network, University of Toronto, Toronto, Ontario, Canada; Department of Medicine, University Health Network, Toronto, Ontario, Canada.

ABSTRACT
Invasive mold infections (IMI) are among the most devastating complications following chemotherapy and hematopoietic stem cell transplantation (HSCT), with high mortality rates. Yet, the molecular basis for human susceptibility to invasive aspergillosis (IA) and mucormycosis remain poorly understood. Herein, we aimed to characterize the immune profile of individuals with hematological malignancies (n = 18) who developed IMI during the course of chemotherapy or HSCT, and compared it to that of hematological patients who had no evidence of invasive fungal infection (n = 16). First, we measured the expression of the pattern recognition receptors pentraxin 3, dectin-1, and Toll-like receptors (TLR) 2 and 4 in peripheral blood of chemotherapy and HSCT recipients with IMI. Compared to hematological controls, individuals with IA and mucormycosis had defective expression of dectin-1; in addition, patients with mucormycosis had decreased TLR2 and increased TLR4 expression. Since fungal recognition via dectin-1 favors T helper 17 responses and the latter are highly dependent on activation of the signal transducer and activator of transcription (STAT) 3, we next used phospho-flow cytometry to measure the phosphorylation of the transcription factors STAT1 and STAT3 in response to interferon-gamma (IFN-γ) and interleukin (IL)-6, respectively. While IFN-γ/STAT1 signaling was similar between groups, naïve T cells from patients with IA, but not those with mucormycosis, exhibited reduced responsiveness to IL-6 as measured by STAT3 phosphorylation. Furthermore, IL-6 increased Aspergillus-induced IL-17 production in culture supernatants from healthy and hematological controls but not in patients with IA. Altogether, these observations suggest an important role for dectin-1 and the IL-6/STAT3 pathway in protective immunity against Aspergillus.

No MeSH data available.


Related in: MedlinePlus

STAT1 and STAT3 phosphorylation in hematological patients with IMI.(a-d) IFN-γ-induced STAT1 phosphorylation and IL-6-induced STAT3 phosphorylation were measured in peripheral blood mononuclear cells using phospho-flow. (a) On the left, representative dot plots for gating of naïve and memory CD4+ T cells are shown; gating of T cells (CD3+) or monocytes (CD33+, not shown) was done on CD45high cells in order to avoid interference of the analysis by potential blasts in leukemic patients with residual disease. On the right, representative histograms of the levels of phosphorylated STAT (pSTAT) proteins in non-stimulated (NS) and cytokine-stimulated cells are shown; baseline pSTAT levels are shown in grey, IFN-γ-induced pSTAT1 is shown in purple and IL-6-induced pSTAT3 is shown in orange. (b) Representative histograms of IL-6 induced pSTAT3 (top row) and IFN-γ-induced pSTAT1 (bottom row) in different patient groups. Y axis corresponds to number of events (i.e. number of naïve CD4+ T cells) and X axis corresponds to fluorescence intensity (i.e. pSTAT-Alexa Fluor 488). (c) Percentage of monocytes (CD45highCD33+ cells), naïve T helper (Th) cells (CD45highCD3+CD4+CD45RO- cells) and memory Th cells (CD45highCD3+CD4+CD45RO+ cells) expressing pSTAT3 (top row) and pSTAT1 (bottom row) in response to IL-6 and IFN-γ, respectively, in healthy controls (blue circles; n = 7), hematological patients with IA (red squares; n = 13) or mucormycosis (black inverted triangles; n = 4) and non-IFI hematological controls (green triangles; n = 15). *p<0.05, **p<0.01 and ***p<0.005 using the unpaired two-tailed Student’s t-test. Data are shown as mean ± s.e.m. (d) Heat map for log2 scale of mean fluorescence intensity (MFI) fold change. Fold change was calculated by dividing the MFI of the cytokine-stimulated sample by that of the unstimulated sample. Heat map color scale is showed in the bottom. Each row on the heat map corresponds to an individual patient or control as indicated by the study ID number on the left. Each column on the heat map corresponds to specific cell type/pSTAT as indicated on the top. Note reduced IL-6-induced pSTAT3 in monocytes (p = 0.01 for IA vs. healthy controls and p<0.05 for IA vs. non-IFI) and naïve CD4+ T cells (p = 0.006 for IA vs. healthy controls and p = 0.04 for IA vs. non-IFI) from patients with IA, using unpaired two-tailed Student’s t-test.
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pone.0123171.g002: STAT1 and STAT3 phosphorylation in hematological patients with IMI.(a-d) IFN-γ-induced STAT1 phosphorylation and IL-6-induced STAT3 phosphorylation were measured in peripheral blood mononuclear cells using phospho-flow. (a) On the left, representative dot plots for gating of naïve and memory CD4+ T cells are shown; gating of T cells (CD3+) or monocytes (CD33+, not shown) was done on CD45high cells in order to avoid interference of the analysis by potential blasts in leukemic patients with residual disease. On the right, representative histograms of the levels of phosphorylated STAT (pSTAT) proteins in non-stimulated (NS) and cytokine-stimulated cells are shown; baseline pSTAT levels are shown in grey, IFN-γ-induced pSTAT1 is shown in purple and IL-6-induced pSTAT3 is shown in orange. (b) Representative histograms of IL-6 induced pSTAT3 (top row) and IFN-γ-induced pSTAT1 (bottom row) in different patient groups. Y axis corresponds to number of events (i.e. number of naïve CD4+ T cells) and X axis corresponds to fluorescence intensity (i.e. pSTAT-Alexa Fluor 488). (c) Percentage of monocytes (CD45highCD33+ cells), naïve T helper (Th) cells (CD45highCD3+CD4+CD45RO- cells) and memory Th cells (CD45highCD3+CD4+CD45RO+ cells) expressing pSTAT3 (top row) and pSTAT1 (bottom row) in response to IL-6 and IFN-γ, respectively, in healthy controls (blue circles; n = 7), hematological patients with IA (red squares; n = 13) or mucormycosis (black inverted triangles; n = 4) and non-IFI hematological controls (green triangles; n = 15). *p<0.05, **p<0.01 and ***p<0.005 using the unpaired two-tailed Student’s t-test. Data are shown as mean ± s.e.m. (d) Heat map for log2 scale of mean fluorescence intensity (MFI) fold change. Fold change was calculated by dividing the MFI of the cytokine-stimulated sample by that of the unstimulated sample. Heat map color scale is showed in the bottom. Each row on the heat map corresponds to an individual patient or control as indicated by the study ID number on the left. Each column on the heat map corresponds to specific cell type/pSTAT as indicated on the top. Note reduced IL-6-induced pSTAT3 in monocytes (p = 0.01 for IA vs. healthy controls and p<0.05 for IA vs. non-IFI) and naïve CD4+ T cells (p = 0.006 for IA vs. healthy controls and p = 0.04 for IA vs. non-IFI) from patients with IA, using unpaired two-tailed Student’s t-test.

Mentions: We next aimed to assess for qualitative defects in CD4+ T cells amongst hematological patients with IMI. T helper (Th) 17 cells play an important role in anti-fungal immunity against Aspergillus [8]. Since dectin-1–mediated fungal recognition favors Th17 differentiation [18,19], we hypothesized that individuals with IMI have defects in the signaling pathways that lead to Th17 differentiation. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways have an important role in the control of innate and adaptive immune responses [20]. Assessment of JAK/STAT phosphorylation by phospho-specific flow cytometric analysis (phospho-flow) [21,22] has been shown to be a useful strategy to identify immune correlates of clinical outcomes in infectious diseases, autoimmunity and cancer [7,23–25]. The interleukin-6 (IL-6)/STAT3 and interferon-gamma (IFN-γ)/STAT1 pathways are critical for the development of Th17 cells [26] and macrophage activation, respectively. Using phospho-flow, we measured IL-6-induced STAT3 and IFN-γ-induced STAT1 phosphorylation in circulating monocytes, naïve and memory CD4+ T cells (Fig 2A). The percentage of naïve CD4+ T cells responding to IL-6, as measured by STAT3 phosphorylation, was significantly reduced in patients with IMI (S2 Fig). This observation was particularly true for patients with IA when compared to healthy and non-IFI hematological controls (54 ± 26 vs. 90 ± 4.72 [p = 0.0003] and 80.4 ± 9.28 [p = 0.004], respectively) (Fig 2B and 2C). The number of memory CD4+ T cells responding to IL-6 was also reduced, though to a less extent, in IA when compared to healthy and non-IFI hematological controls (Fig 2C). Patients with mucormycosis had reduced numbers of monocytes and memory CD4+ T cells responding to IL-6, as compared to healthy controls (Fig 2C). Monocyte and lymphocyte responsiveness to IFN-γ was similar across all study groups (Fig 2C). In addition to the reduced percentage of naïve CD4+ T cells responding to IL-6 (Fig 2C), the magnitude of IL-6-induced STAT3 phosphorylation on naïve CD4+ T cells was also significantly reduced in patients with IA (Fig 2D) but not in those with mucormycosis (fluorescence intensity fold change: 2.66 ± 0.74 vs. 6.28 ± 2.66 [p = 0.02] and 3.54 ± 1.31 [p = 0.04] for IA vs. healthy and non-IFI hematological controls, respectively). Similarly, IA cases exhibited defective monocyte responsiveness to IL-6, as measured by STAT3 phosphorylation (fluorescence intensity fold change: 2.40 ± 1.08), when compared to healthy and non-IFI controls (4.67 ±1.81 [p = 0.03] and 3.50 ± 1.23 [p = 0.04], respectively) (Fig 2D). These findings are consistent with the increased risk of IA previously reported in patients with autosomal dominant STAT3 deficiency [27,28].


Impaired T cell responsiveness to interleukin-6 in hematological patients with invasive aspergillosis.

Camargo JF, Bhimji A, Kumar D, Kaul R, Pavan R, Schuh A, Seftel M, Lipton JH, Gupta V, Humar A, Husain S - PLoS ONE (2015)

STAT1 and STAT3 phosphorylation in hematological patients with IMI.(a-d) IFN-γ-induced STAT1 phosphorylation and IL-6-induced STAT3 phosphorylation were measured in peripheral blood mononuclear cells using phospho-flow. (a) On the left, representative dot plots for gating of naïve and memory CD4+ T cells are shown; gating of T cells (CD3+) or monocytes (CD33+, not shown) was done on CD45high cells in order to avoid interference of the analysis by potential blasts in leukemic patients with residual disease. On the right, representative histograms of the levels of phosphorylated STAT (pSTAT) proteins in non-stimulated (NS) and cytokine-stimulated cells are shown; baseline pSTAT levels are shown in grey, IFN-γ-induced pSTAT1 is shown in purple and IL-6-induced pSTAT3 is shown in orange. (b) Representative histograms of IL-6 induced pSTAT3 (top row) and IFN-γ-induced pSTAT1 (bottom row) in different patient groups. Y axis corresponds to number of events (i.e. number of naïve CD4+ T cells) and X axis corresponds to fluorescence intensity (i.e. pSTAT-Alexa Fluor 488). (c) Percentage of monocytes (CD45highCD33+ cells), naïve T helper (Th) cells (CD45highCD3+CD4+CD45RO- cells) and memory Th cells (CD45highCD3+CD4+CD45RO+ cells) expressing pSTAT3 (top row) and pSTAT1 (bottom row) in response to IL-6 and IFN-γ, respectively, in healthy controls (blue circles; n = 7), hematological patients with IA (red squares; n = 13) or mucormycosis (black inverted triangles; n = 4) and non-IFI hematological controls (green triangles; n = 15). *p<0.05, **p<0.01 and ***p<0.005 using the unpaired two-tailed Student’s t-test. Data are shown as mean ± s.e.m. (d) Heat map for log2 scale of mean fluorescence intensity (MFI) fold change. Fold change was calculated by dividing the MFI of the cytokine-stimulated sample by that of the unstimulated sample. Heat map color scale is showed in the bottom. Each row on the heat map corresponds to an individual patient or control as indicated by the study ID number on the left. Each column on the heat map corresponds to specific cell type/pSTAT as indicated on the top. Note reduced IL-6-induced pSTAT3 in monocytes (p = 0.01 for IA vs. healthy controls and p<0.05 for IA vs. non-IFI) and naïve CD4+ T cells (p = 0.006 for IA vs. healthy controls and p = 0.04 for IA vs. non-IFI) from patients with IA, using unpaired two-tailed Student’s t-test.
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pone.0123171.g002: STAT1 and STAT3 phosphorylation in hematological patients with IMI.(a-d) IFN-γ-induced STAT1 phosphorylation and IL-6-induced STAT3 phosphorylation were measured in peripheral blood mononuclear cells using phospho-flow. (a) On the left, representative dot plots for gating of naïve and memory CD4+ T cells are shown; gating of T cells (CD3+) or monocytes (CD33+, not shown) was done on CD45high cells in order to avoid interference of the analysis by potential blasts in leukemic patients with residual disease. On the right, representative histograms of the levels of phosphorylated STAT (pSTAT) proteins in non-stimulated (NS) and cytokine-stimulated cells are shown; baseline pSTAT levels are shown in grey, IFN-γ-induced pSTAT1 is shown in purple and IL-6-induced pSTAT3 is shown in orange. (b) Representative histograms of IL-6 induced pSTAT3 (top row) and IFN-γ-induced pSTAT1 (bottom row) in different patient groups. Y axis corresponds to number of events (i.e. number of naïve CD4+ T cells) and X axis corresponds to fluorescence intensity (i.e. pSTAT-Alexa Fluor 488). (c) Percentage of monocytes (CD45highCD33+ cells), naïve T helper (Th) cells (CD45highCD3+CD4+CD45RO- cells) and memory Th cells (CD45highCD3+CD4+CD45RO+ cells) expressing pSTAT3 (top row) and pSTAT1 (bottom row) in response to IL-6 and IFN-γ, respectively, in healthy controls (blue circles; n = 7), hematological patients with IA (red squares; n = 13) or mucormycosis (black inverted triangles; n = 4) and non-IFI hematological controls (green triangles; n = 15). *p<0.05, **p<0.01 and ***p<0.005 using the unpaired two-tailed Student’s t-test. Data are shown as mean ± s.e.m. (d) Heat map for log2 scale of mean fluorescence intensity (MFI) fold change. Fold change was calculated by dividing the MFI of the cytokine-stimulated sample by that of the unstimulated sample. Heat map color scale is showed in the bottom. Each row on the heat map corresponds to an individual patient or control as indicated by the study ID number on the left. Each column on the heat map corresponds to specific cell type/pSTAT as indicated on the top. Note reduced IL-6-induced pSTAT3 in monocytes (p = 0.01 for IA vs. healthy controls and p<0.05 for IA vs. non-IFI) and naïve CD4+ T cells (p = 0.006 for IA vs. healthy controls and p = 0.04 for IA vs. non-IFI) from patients with IA, using unpaired two-tailed Student’s t-test.
Mentions: We next aimed to assess for qualitative defects in CD4+ T cells amongst hematological patients with IMI. T helper (Th) 17 cells play an important role in anti-fungal immunity against Aspergillus [8]. Since dectin-1–mediated fungal recognition favors Th17 differentiation [18,19], we hypothesized that individuals with IMI have defects in the signaling pathways that lead to Th17 differentiation. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways have an important role in the control of innate and adaptive immune responses [20]. Assessment of JAK/STAT phosphorylation by phospho-specific flow cytometric analysis (phospho-flow) [21,22] has been shown to be a useful strategy to identify immune correlates of clinical outcomes in infectious diseases, autoimmunity and cancer [7,23–25]. The interleukin-6 (IL-6)/STAT3 and interferon-gamma (IFN-γ)/STAT1 pathways are critical for the development of Th17 cells [26] and macrophage activation, respectively. Using phospho-flow, we measured IL-6-induced STAT3 and IFN-γ-induced STAT1 phosphorylation in circulating monocytes, naïve and memory CD4+ T cells (Fig 2A). The percentage of naïve CD4+ T cells responding to IL-6, as measured by STAT3 phosphorylation, was significantly reduced in patients with IMI (S2 Fig). This observation was particularly true for patients with IA when compared to healthy and non-IFI hematological controls (54 ± 26 vs. 90 ± 4.72 [p = 0.0003] and 80.4 ± 9.28 [p = 0.004], respectively) (Fig 2B and 2C). The number of memory CD4+ T cells responding to IL-6 was also reduced, though to a less extent, in IA when compared to healthy and non-IFI hematological controls (Fig 2C). Patients with mucormycosis had reduced numbers of monocytes and memory CD4+ T cells responding to IL-6, as compared to healthy controls (Fig 2C). Monocyte and lymphocyte responsiveness to IFN-γ was similar across all study groups (Fig 2C). In addition to the reduced percentage of naïve CD4+ T cells responding to IL-6 (Fig 2C), the magnitude of IL-6-induced STAT3 phosphorylation on naïve CD4+ T cells was also significantly reduced in patients with IA (Fig 2D) but not in those with mucormycosis (fluorescence intensity fold change: 2.66 ± 0.74 vs. 6.28 ± 2.66 [p = 0.02] and 3.54 ± 1.31 [p = 0.04] for IA vs. healthy and non-IFI hematological controls, respectively). Similarly, IA cases exhibited defective monocyte responsiveness to IL-6, as measured by STAT3 phosphorylation (fluorescence intensity fold change: 2.40 ± 1.08), when compared to healthy and non-IFI controls (4.67 ±1.81 [p = 0.03] and 3.50 ± 1.23 [p = 0.04], respectively) (Fig 2D). These findings are consistent with the increased risk of IA previously reported in patients with autosomal dominant STAT3 deficiency [27,28].

Bottom Line: While IFN-γ/STAT1 signaling was similar between groups, naïve T cells from patients with IA, but not those with mucormycosis, exhibited reduced responsiveness to IL-6 as measured by STAT3 phosphorylation.Furthermore, IL-6 increased Aspergillus-induced IL-17 production in culture supernatants from healthy and hematological controls but not in patients with IA.Altogether, these observations suggest an important role for dectin-1 and the IL-6/STAT3 pathway in protective immunity against Aspergillus.

View Article: PubMed Central - PubMed

Affiliation: Transplant Infectious Diseases, Multi-Organ Transplant Program, University Health Network, University of Toronto, Toronto, Ontario, Canada; Department of Medicine, University Health Network, Toronto, Ontario, Canada.

ABSTRACT
Invasive mold infections (IMI) are among the most devastating complications following chemotherapy and hematopoietic stem cell transplantation (HSCT), with high mortality rates. Yet, the molecular basis for human susceptibility to invasive aspergillosis (IA) and mucormycosis remain poorly understood. Herein, we aimed to characterize the immune profile of individuals with hematological malignancies (n = 18) who developed IMI during the course of chemotherapy or HSCT, and compared it to that of hematological patients who had no evidence of invasive fungal infection (n = 16). First, we measured the expression of the pattern recognition receptors pentraxin 3, dectin-1, and Toll-like receptors (TLR) 2 and 4 in peripheral blood of chemotherapy and HSCT recipients with IMI. Compared to hematological controls, individuals with IA and mucormycosis had defective expression of dectin-1; in addition, patients with mucormycosis had decreased TLR2 and increased TLR4 expression. Since fungal recognition via dectin-1 favors T helper 17 responses and the latter are highly dependent on activation of the signal transducer and activator of transcription (STAT) 3, we next used phospho-flow cytometry to measure the phosphorylation of the transcription factors STAT1 and STAT3 in response to interferon-gamma (IFN-γ) and interleukin (IL)-6, respectively. While IFN-γ/STAT1 signaling was similar between groups, naïve T cells from patients with IA, but not those with mucormycosis, exhibited reduced responsiveness to IL-6 as measured by STAT3 phosphorylation. Furthermore, IL-6 increased Aspergillus-induced IL-17 production in culture supernatants from healthy and hematological controls but not in patients with IA. Altogether, these observations suggest an important role for dectin-1 and the IL-6/STAT3 pathway in protective immunity against Aspergillus.

No MeSH data available.


Related in: MedlinePlus