Coinfection with Leishmania major and Staphylococcus aureus enhances the pathologic responses to both microbes through a pathway involving IL-17A

Abstract

Cutaneous leishmaniasis (CL) is a parasitic disease causing chronic, ulcerating skin lesions. Most humans infected with the causative Leishmania protozoa are asymptomatic. Leishmania spp. are usually introduced by sand flies into the dermis of mammalian hosts in the presence of bacteria from either the host skin, sand fly gut or both. We hypothesized that bacteria at the dermal inoculation site of Leishmania major will influence the severity of infection that ensues. A C57BL/6 mouse ear model of single or coinfection with Leishmania major, Staphylococcus aureus, or both showed that single pathogen infections caused localized lesions that peaked after 2-3 days for S. aureus and 3 weeks for L. major infection, but that coinfection produced lesions that were two-fold larger than single infection throughout 4 weeks after coinfection. Coinfection increased S. aureus burdens over 7 days, whereas L. major burdens (3, 7, 28 days) were the same in singly and coinfected ears. Inflammatory lesions throughout the first 4 weeks of coinfection had more neutrophils than did singly infected lesions, and the recruited neutrophils from early (day 1) lesions had similar phagocytic and NADPH oxidase capacities. However, most neutrophils were apoptotic, and transcription of immunomodulatory genes that promote efferocytosis was not upregulated, suggesting that the increased numbers of neutrophils may, in part, reflect defective clearance and resolution of the inflammatory response. In addition, the presence of more IL-17A-producing γδ and non-γδ T cells in early lesions (1-7 days), and L. major antigen-responsive Th17 cells after 28 days of coinfection, with a corresponding increase in IL-1β, may recruit more naïve neutrophils into the inflammatory site. Neutralization studies suggest that IL-17A contributed to an enhanced inflammatory response, whereas IL-1β has an important role in controlling bacterial replication. Taken together, these data suggest that coinfection of L. major infection with S. aureus exacerbates disease, both by promoting more inflammation and neutrophil recruitment and by increasing neutrophil apoptosis and delaying resolution of the inflammatory response. These data illustrate the profound impact that coinfecting microorganisms can exert on inflammatory lesion pathology and host adaptive immune responses.

Fig 1. Sequential, prior, or simultaneous introduction of S. aureus during L. major infection.

C57BL/6 mice were inoculated in ears with buffer (PBS), either WT Newman S. aureus or S. aureus LAC::lux (Sa), L. major (Lm) or S. aureus and L. major (L+S) with different timing or body sites of bacterial versus parasitic infection. Lesion sizes were measured throughout infection (panels A, E, G). Lm parasite loads were measured in the same mice at the end of experiments (panels B, F, H). (A) Simultaneous intradermal coinfection (L+S) in the C57BL/6 mouse ear pinna results in significantly greater lesion size than either organism alone. Data represent the mean ± SEM lesion volumes at different time points in three independent experiments, each with 4–5 mice/group. Asterisks (*) represent significance between Sa and L+S groups. Crosshairs (+) represent significance between Lm and L+S groups. (B) Parasite loads corresponding to single or coinfections in panel A. S. aureus infections were done using WT Newman strain S. aureus. Lm burdens were measured by qPCR of DNA from infected ears 28 days p.i. Data represent the mean ± SEM of three independent experiments, each with 4–5 mice/group. (C) In vivo imaging of Sa loads in single or coinfected mice: S. aureus LAC::lux was inoculated alone or with Lm. Bioluminescence corresponding to the load of live Sa was measured by in vivo imaging daily for 7 days. Data represent the mean ± SEM of two independent experiments, each with 5–10 mice/group. (D) (Left panel) Total Sa burden was measured by qPCR of DNA extracted from 28 days p.i. ears for one of the experiments in panel C. Data show the mean ± SD S. aureus genome equivalents in 5 mice/group. (Right panel) Live Sa burdens were also measured by counting colony-forming units (CFUs) after overnight growth from ear homogenates. Data represent the mean ± SEM CFU in the two independent experiments, each with 4–5 mice/group. (E) L. major infection prior to secondary S. aureus infection: mice were injected with Lm or phosphate buffered saline (PBS) intradermally in the ear on day 0. On day 21, the infection site was injected with either S. aureus to model superinfection of an established lesion (Lm-Sa), or PBS as a control (Lm-PBS). Data represent mean ± SEM lesion sizes at each time point from three independent experiments, each with 5 mice/group. Asterisks (*) indicate significance between PBS-Sa and Lm-Sa groups at all times beyond 21 days. (F) Parasite loads: Lm burdens 28 days p.i., corresponding to the Lm-PBS control or Lm-Sa sequential infection groups in panel A, were measured by qPCR in total ear DNA. Parasite burdens are expressed as Lm genome equivalents/ear. Data represent mean ± SEM from three independent experiments, each with 5 mice/group. (G) S. aureus infection prior to or simultaneous with L. major infection. (i) Prior: to model the importance of preexisting skin bacteria on lesion development, mice were injected with Sa or PBS intradermally in the right ear on day -10. On day 0, either the same ear or the opposite (left) ear was injected intradermally with Lm (closed symbols). (ii) Simultaneous: on day 0, groups of naïve mice were injected simultaneously with Lm and Sa (open symbols) in the same ear (L+S), or Sa in the right ear and Lm in the left ear (Sa(R)+Lm(L)). Lesion volume measurements are shown. Asterisks (*) represent significance between L+S coinfection group compared to all other groups. Data represented as the mean ± SD of one experiment with 5 mice/group. (H) L. major parasite burdens corresponding to panel C: despite significantly different lesion sizes, parasite loads were not significantly different between mice coinfected with L+S in the same or opposite ears, or by Sa prior to Lm infection. Data shown as the mean ± SD of one experiment with 5 mice/group. Unless otherwise indicated, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant by one-way (H) or two-way (A, C, E, and G) ANOVA with Tukey’s post-test, or Student’s t-test (B, D, and F).

Fig 2. L. major-S. aureus coinfection results in greater neutrophil infiltration than L. major alone.

(A) Low-powered image (100x) of paraffin-embedded hematoxylin and eosin-stained sections of ears from mice intradermally injected with S. aureus (Sa), L. major (Lm), or co-inoculated with both (L+S) 28 days p.i. (B) High-powered image (1000x) of histological section from coinfected ear. White bar represents 10 μm. (C) Histological inflammatory cell scores for lymphocytes and histiocytes or (D) neutrophils from ears 28 days p.i. were calculated as the average score of four high-powered (400x) fields per ear. (E) Numbers of neutrophils (PMNs) (CD45⁺ CD11b⁺ Ly6G^hi Ly6C^int), inflammatory monocytes (MN) (CD45⁺ CD11b⁺ Ly6G^- Ly6C^hi), and myeloid dendritic cells (DC) (CD45⁺ CD11b⁺ CD11c⁺), shown as absolute cell numbers in samples derived from inoculated ears at days 1 and 7 p.i. was determined by flow cytometry. (F) Numbers of γδ (CD45⁺ Thy1.2⁺ γδ TCR⁺) and non-γδ (CD45⁺ Thy1.2⁺ γδ TCR^-) T cell numbers from the ears at days 1 and 7 p.i. was determined by analyzing single cells suspensions from whole ear samples by flow cytometry. Data are shown as the mean ± SEM of three separate experiments, each with 4–5 mice/group (A, B, C, and D), or as the mean ± SD of 5 mice/group in one of three representative experiments (E and F). *p < 0.05, **p < 0.01, ***p < 0.0001, ns = not significant by one-way ANOVA (C and D) or two-way ANOVA (E and F) with Tukey post-test for multiple comparisons.

Fig 3. Neutrophils in L. major-S. aureus coinfected lesions can phagocytose pathogens and have functional NADPH oxidase but are mostly apoptotic.

(A) Percentage of myeloid cells that are neutrophils from mice injected intradermally in the ear with phosphate buffered saline (PBS), L. major IA-2 (Lm), S. aureus Newman (Sa), or coinfected with L. major and S. aureus (L+S) for one day. (B) Numbers of polymorphonuclear cells (PMNs) positive for S. aureus LAC GFP or L. major IA-2 luc mCherry in singly infected versus coinfected lesions. (C) Dihydrorhodamine (DHR) geometric mean fluorescence intensity (GMFI) of neutrophils stimulated with PMA ex vivo in the presence of DHR to assess NADPH oxidase activity. (D) Annexin V (AnnV) and propidium iodide (PI) staining of neutrophils from PBS, S. aureus, L. major, or coinfected lesions. (E) Percentage of apoptotic (Annexin V⁺) versus live (AnnV^-PI^-) neutrophils from day 1 p.i. PBS, S. aureus, L. major, or coinfected ears. Data are represented as the mean ± SEM of two replicate experiments each with 5 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant by Student’s t test (B), or one-way (A, C) or two-way ANOVA (D) with Tukey’s post-test.

Fig 4. Immune cells from L. major-S. aureus coinfected mice produce similar levels of Th1- and Th2-type cytokines, but more IL-17A than singly infected mice in response to L. major antigen.

Draining lymph nodes from mice day 28 p.i. were re-stimulated with live L. major promastigote antigen for 72 hours and cytokine levels in culture supernatants were assayed by multiplex ELISA. Data represent the mean ± SEM of three replicate experiments, each with 4–5 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant by one-way ANOVA with Tukey’s post-test. PBS = phosphate buffered saline, Sa = S. aureus, Lm = L. major, L+S = L. major + S. aureus.

Fig 5. L. major and S. aureus coinfection results in elevated levels of immune cells producing proinflammatory cytokines.

Ears and draining lymph nodes of mice were homogenized, cultured for 4–5 hours with BFA, PMA, and ionomycin, and stained for surface markers, intracellular IL-17A or IFNγ, and analyzed by flow cytometry. Stained cells were gated on surface markers followed by intracellular cytokines. (A) IL-17A⁺ γδ (CD45⁺ Thy1.2⁺ γδ TCR⁺) and non-γδ (CD45⁺ Thy1.2⁺ γδ TCR^-) T cell numbers from the ears at days 1, 7, and 28 p.i. (B) Numbers of IL-17A⁺ cells co-staining with CD11b from ears 1 day p.i. (C) Numbers of IL-17A⁺ cells co-staining CD45⁺ CD11b⁺ Ly6G^hi Ly6C^int (PMN), CD45⁺ CD11b⁺ Ly6G^- Ly6C^hi (MN), or CD45⁺ CD11b⁺ CD11c⁺ (DC) from the ears at 1 day p.i. (D) Numbers of IL-17A⁺ CD4 (CD11b^- Thy1.2⁺ CD4⁺ CD8^-) and CD8 (CD11b^- Thy1.2⁺ CD4^- CD8⁺) T cells from draining lymph nodes at day 28 p.i. (E) Numbers of IFNγ⁺ γδ (CD11b^- Thy1.2⁺ γδ TCR⁺) and non-γδ (CD11b^- Thy1.2⁺ γδ TCR^-) T cells from the ears at days 1, 7, and 28 p.i. Data at day 7 and CD11b⁺ cell data (panels A, B, C, & E) are shown as the mean ± SEM of two pooled experiments, each with 3–6 mice/group. T cell data at day 1 and 28 (panels A, D, & E) are representative of one experiment and shown as the mean ± SD of 5 mice/group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (B) or two-way ANOVA (A, C, D, and E) with Tukey’s post-test. PBS = phosphate buffered saline, Sa = S. aureus, Lm = L. major, L+S = L. major + S. aureus.

Fig 6. L. major-S. aureus coinfection results in increased pro-IL-1β expressing myeloid cells, higher IL-1β levels, and greater IL-23 gene expression in infected tissues.

Infected mice ears were homogenized and (A) cultured for 4–5 hours with BFA, PMA, and ionomycin, stained for surface markers and intracellular pro-IL1β, and analyzed by flow cytometry for (B) numbers of pro-IL-1β⁺ cells co-staining CD45⁺ CD11b⁺ Ly6G^hi Ly6C^int (PMN), CD45⁺ CD11b⁺ Ly6G^- Ly6C^hi cells (MN), or CD45⁺ CD11b⁺ CD11c⁺ (DC) from the ears at days 1 and 7 p.i. (C) Supernatants from homogenized ear tissues were analyzed by ELISA for IL-1β concentration. (D) IL-6 gene expression from ears 3 days p.i. (E) IL-23 gene expression from ears 3 and 7 days p.i. Data from days 1 and 3 (A-C) and panels D & E represent one experiment and are shown as the mean ± SD of 5 mice/group. Data from day 7 (A & B) from two replicate experiments are shown as the mean ± SEM of 3–6 mice/group. *p < 0.05, **p < 0.0001 by one-way ANOVA with Tukey’s post-test. PBS = phosphate buffered saline, Sa = S. aureus, Lm = L. major, L+S = L. major + S. aureus.

Fig 7. Coinfection with S. aureus MNPE, a USA200 TSST⁺ clinical strain, recapitulates early L. major-S. aureus Newman lesion exacerbation and cytokine production.

Mice were injected intradermally in the ear with phosphate buffered saline (PBS), L. major (Lm), S. aureus Newman (Sa Newman), S. aureus MNPE (Sa MNPE) or co-inoculated with L. major and either S. aureus strain (L+S Newman or L+S MNPE) and ear lesion volume was measured for 7 days. On day 7 p.i., single cell suspensions from the ears were obtained, treated with BFA, PMA, and ionomycin for 4–5 hours, stained for surface markers and intracellular cytokines, and analyzed by flow cytometry. (A) Lesion volume of ears coinfected with L. major and S. aureus MNPE compared to those coinfected with S. aureus Newman MSSA. (B) IL-17A⁺ or IFNγ⁺ γδ (CD45⁺ Thy1.2⁺ γδ TCR⁺) and non-γδ (CD45⁺ Thy1.2⁺ γδ TCR^-) T cell numbers in ears. Data represent the mean ± SD of one experiment with 5–6 mice/group. Lesion size data are representative of two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA with Tukey’s post-test.

Fig 8. Anti-IL-17A neutralizing antibodies partially ameliorate early coinfection lesion exacerbation while anti-IL-1β antibodies further exacerbate lesions.

Mice were injected intraperitoneally with 0.5 mg neutralizing antibody or isotype control antibody one day prior to intradermal ear injection with L. major (Lm) or L. major and S. aureus (L+S). Neutralizing antibody was administered one day prior to infection and every three days for 9 days. Ear lesion volumes during coinfection in mice treated with (A) anti-IL-17A or MOPC-21 isotype antibodies, or (B) anti-IL-1β or polyclonal Armenian hamster IgG isotype antibodies are shown. Data represent the mean ± SEM of two independent experiments, each with 5 mice per group. IgG L+S versus anti-IL-17A L+S or anti-IL-1β L+S comparisons: *p < 0.01, **p < 0.001, ***p < 0.0001 by two-way ANOVA with Tukey’s post-test. (C) Bacterial load by in vivo bioluminescence imaging of mice treated with anti-IL-1β or polyclonal Armenian hamster IgG isotype antibodies and infected with 10⁴ CFU S. aureus LAC::lux alone. Data represent the mean ± SD of one experiment with 5 mice per group. ***p < 0.0001 by two-way ANOVA with Tukey’s post-test.

Fig 9. Proposed mechanism of L. major and S. aureus coinfection lesion exacerbation.

When an infected sand fly takes a blood meal, it egests L. major into the skin, and S. aureus from the skin microbiota or from the sand fly gut also enters the skin. These organisms get taken up by host macrophages and neutrophils and promote the production of proinflammatory IL-1β and IL-23 by myeloid cells. The presence of L. major allows for enhanced S. aureus growth during the early stages of infection. Although it is reported that IL-1β and IL-23 promote the production of IL-17A from γδ and non-γδ T cells, and the differentiation of naïve T helper cells into Th17 cells, IL-1β may have an important role in control of S. aureus growth and lesion pathology related to bacterial burden. Elevated IL-17A expression in correlated with more neutrophils at the coinfected lesion site that were mostly apoptotic in a setting of downregulated efferocytosis. The IL-17A pathway may be largely responsible for the exacerbation of coinfected lesions.