Th1-Th17 cells mediate protective adaptive immunity against Staphylococcus aureus and Candida albicans infection in mice

Abstract

We sought to define protective mechanisms of immunity to Staphylococcus aureus and Candida albicans bloodstream infections in mice immunized with the recombinant N-terminus of Als3p (rAls3p-N) vaccine plus aluminum hydroxide (Al(OH(3)) adjuvant, or adjuvant controls. Deficiency of IFN-gamma but not IL-17A enhanced susceptibility of control mice to both infections. However, vaccine-induced protective immunity against both infections required CD4+ T-cell-derived IFN-gamma and IL-17A, and functional phagocytic effectors. Vaccination primed Th1, Th17, and Th1/17 lymphocytes, which produced pro-inflammatory cytokines that enhanced phagocytic killing of both organisms. Vaccinated, infected mice had increased IFN-gamma, IL-17, and KC, increased neutrophil influx, and decreased organism burden in tissues. In summary, rAls3p-N vaccination induced a Th1/Th17 response, resulting in recruitment and activation of phagocytes at sites of infection, and more effective clearance of S. aureus and C. albicans from tissues. Thus, vaccine-mediated adaptive immunity can protect against both infections by targeting microbes for destruction by innate effectors.

Figure 1. CD4 derived IFN-γ is required for vaccine protection.

Wild type Balb/c mice were infected with 2×10⁵ C. albicans SC5314 or 2×10⁷ S. aureus LAC; IFN-γ deficient mice on a Balb/c background were infected with 10⁵ C. albicans or 10⁷ S. aureus. A) Wild type, n = 8 mice per group, or IFN-γ deficient mice, n = 7 mice per group, were vaccinated and infected iv via the tail-vein 2 weeks after the booster dose. *p<0.05 for wild type vaccinated vs. control by Log Rank test. B) CD4+ T cells, 5×10⁶, from vaccinated or control mice were adoptively transferred iv to recipient mice, n = 8 mice per group, 24 h prior to infection. *p<0.05 for wild type donor cells vaccinated vs. control by Log Rank test.

Figure 2. Chemotherapy-induced neutropenia ablated vaccine induced protection.

Sixteen Balb/c mice per group were vaccinated with rAls3p-N plus AlOH₃ or AlOH₃ alone, and boosted three weeks later. Two weeks after the boost, half the mice were treated with cyclophosphamide. Two days later the mice were infected with C. albicans SC5314, 1.5×10⁵, C. albicans 15563, 7×10⁵, or MRSA LAC, 1.5×10⁷. *p<0.05 for vaccinated vs. control by Log Rank test.

Figure 3. Phagocyte superoxide production is required for vaccine protection.

N = 8 mice per group. Wild type C57BL/6 mice were infected with 1.5×10⁵ C. albicans SC5314 or 2×10⁷ S. aureus LAC; gp91^phox−/− mice on a C57BL/6 background were infected with 1.5×10³ C. albicans or 10⁷ S. aureus. A) Mice were vaccinated and infected as above. *p<0.05 for wild type vaccinated vs. control by Log Rank test. B) CD4+ T cells, 5×10⁶, from vaccinated or control, wild type or gp91^phox−/− mice were cross-adoptively transferred iv to recipient mice 24 h prior to infection–wild type cells transferred to gp91^phox−/− mice, and visa versa. *p<0.05 for wild type donor cells vaccinated vs. control by Log Rank test.

Figure 4. CD4+ T cell derived IL-17A was required for vaccine protection.

A) Balb/c or IL-17A deficient mice on a Balb/c background (n = 8 per group) were vaccinated with rAls3p-N plus Alhydrogel or Alhydrogel alone, with a boost at 3 weeks. Two weeks after the boost, all mice were infected with 2.5×10⁵ C. albicans SC5314 or 2×10⁷ S. aureus LAC. B) Balb/c mice or IL-17A deficient mice on a Balb/c background, n = 8 per group, were vaccinated with rAls3p-N plus Alhydrogel or Alhydrogel alone. Two weeks after the boost, splenic and lymph node CD4+ T cells, 5×10⁶, from vaccinated or control, wild type or IL-17A-deficient mice were cross-adoptively transferred iv to recipient mice, wild type donor to IL-17A deficient recipient, IL-17A donor to wild type recipient, 24 h prior to infection with C. albicans SC5314, 2.5×10⁵ inoculum. *p<0.05 for wild type donor vaccinated vs. control by Log Rank test.

Figure 5. Vaccination primed lymphocytes to produce pro-inflammatory, Th1/Th17 cytokines which enhanced phagocytic killing of both organisms.

A) Balb/c mice, n = 8 per group, were vaccinated with rAls3p-N plus Alhydrogel or Alhydrogel alone = Control. Two weeks after the boost splenocytes and cervical and axillary lymph node cells were harvested and incubated for 5 days with rAls3p-N. Supernatant was harvested for determination of cytokine levels. Median and interquartile ranges are shown. *p<0.03 vs. Control. B) RAW murine macrophage cells or freshly harvested murine neutrophils were primed with the above supernatants for four hours prior to incubation for one additional hour with C. albicans SC5314 (20∶1 RAW to C. albicans; 10∶1 neutrophils to C. albicans) or S. aureus LAC (5∶1 RAW to S. aureus; 10∶1 neutrophils to S. aureus). The culture wells were overlaid with agar and colonies counted the following day. Percent killing reflects reduction in colony forming units compared to number of organisms added to the wells. Median and interquartile ranges are graphed. *p<0.05 for immune vs. control supernatant.

Figure 6. Vaccination primed Th1, Th17, and Th1/17 cells in draining lymph nodes.

Balb/c mice, n = 4 per group, were vaccinated with rAls3p-N plus Alhydrogel or Alhydrogel alone = Control. Two weeks after the boost cervical and axillary lymph node cells were harvested and incubated for 5 days with rAls3p-N, followed by 6 h with PMA/ionomycin and monensin. Cells were fixed, permeabilized, and stained for CD4, CCR6, IFN-γ, or IL-17. Intracellular cytokine analysis was conducted by flow cytometry. “All Lymphocytes” were gated based on size by FSC and density by SSC; CD4+ and CCR6+ lymphocytes were further gated from the “All Lymphocyte” population by fluorescence signal indicating surface expression of these markers. Median and interquartile ranges are shown. *P<0.05 vs. Control.

Figure 7. Vaccination reduced infectious burden and stimulated neutrophil influx by MPO and pro-inflammatory cytokine levels in kidneys.

Balb/c mice, n = 8 per group, were vaccinated with rAls3p-N plus Alhydrogel or Alhydrogel alone. Two weeks after the boost, mice were infected iv with C. albicans SC5314, 2×10⁵, or S. aureus LAC, 3×10⁷. Four days after infection, individually marked kidneys (primary target organ of infection for both models) were harvested, homogenized, and quantitatively cultured (A). MPO levels (B) and cytokine levels (C) in organ homogenates were measured by ELISA. MPO levels are shown both as raw values in ng/g of kidney tissue, and also normalized to burden of infection in each individually marked organ in fg/g tissue/CFU per organ. Cytokine levels are shown normalized to organ CFU burden. Median and interquartile ranges are shown. *p<0.05 vs. Control by Mann Whitney U test.

Figure 8. Unvaccinated mice had less neutrophil influx relative to fungal and bacterial burden than vaccinated mice.

Representative histopathological sections from kidneys from 2 mice per group are shown. Control mice infected with C. albicans had multiple abscesses with visible hyphae and pseudohyphae throughout the kidneys. Vaccinated mice infected with C. albicans had abscesses, but with far less fungus visible. Numerous abscesses were seen in both vaccinated and control mice infected with S. aureus. However, overall the abscesses in the control mice infected with S. aureus had considerably more bacteria by gram stain than the abscesses in the vaccinated mice. Sections were stained by PAS (for C. albicans) or H&E (to show the neutrophil influx and the extent of tissue necrosis) and Gram stain (to show S. aureus dark purple clusters). Magnification = 1000×.