SIGNR1-negative red pulp macrophages protect against acute streptococcal sepsis after Leishmania donovani-induced loss of marginal zone macrophages

Abstract

Marginal zone macrophages in the murine spleen play an important role in the capture of blood-borne pathogens and are viewed as an essential component of host defense against the development of pneumococcal sepsis. However, we and others have previously described the loss of marginal zone macrophages associated with the splenomegaly that follows a variety of viral and protozoal infections; this finding raises the question of whether these infected mice would become more susceptible to secondary pneumococcal infection. Contrary to expectations, we found that mice lacking marginal zone macrophages resulting from Leishmania donovani infection have increased resistance to Streptococcus pneumoniae type 3 and do not develop sepsis. Using biophotonic imaging, we observed that pneumococci are rapidly trapped in the spleens of L. donovani-infected mice. By selective depletion studies using clodronate liposomes, depleting monoclonal antibodies specific for Ly6C/G and Ly6G, and CD11c-DTR mice, we show that the enhanced early resistance in L. donovani-infected mice is entirely due to the activity of SIGNR1(-) red pulp macrophages. Our data demonstrate, therefore, that the normal requirement for SIGNR1(+) marginal zone macrophages to protect against a primary pneumococcal infection can, under conditions of splenomegaly, be readily compensated for by activated red pulp macrophages.

Figure 1

Progression of acute S. pneumoniae Xen10 infection in control and L. donovani-infected mice. A: Control mice (left panels) or mice at day 42 after L. donovani infection (right panels) were challenged with bioluminescent S. pneumoniae Xen10 i.v. and monitored for total body luminescence. Colored scale bars denote luminescence (photons/second/cm²/sr). Note the differing scale for top and bottom panels. Red squares and ellipses in top panels indicate areas used to measure torso and splenic luminescence, respectively, in subsequent figures. Images are representative of all imaging experiments. B: Graph shows survival of previously naive mice (n = 30; solid line) and L. donovani-infected mice (n = 20; broken line) over the first 20 hours after an i.v. challenge with S. pneumoniae Xen10. Survival is significantly enhanced in L. donovani-infected mice (P < 0.0001; two-tailed log-rank test). C: Control and L. donovani-infected mice were infected i.v. with S. pneumoniae Xen10. Bioluminescence of the spleen and torso of individual mice were measured at various time points after infection. Top: bioluminescence of individual control (closed circles) and L. donovani-infected (open circles) mice. Bottom: mean ± SD at each time point. *P < 0.05 by Student’s t-test. Graphs are representative of three individual experiments.

Figure 2

Serum from L. donovani-infected mice does not enhance clearance of S. pneumoniae. A and B: Previously naive mice were given serum from naive control mice or from mice at day 42 of L. donovani infection. At 4 hours after i.v. serum transfer each group was challenged i.v. with S. pneumoniae Xen10, and bioluminescence was measured at various time points. Images are representative of bioluminescence observed at 4 hours after infection in mice given (A) naive serum and (B) serum from L. donovani-infected donors. C and D: Graphs show mean ± SD bioluminescence in the spleen and torso of mice given naive serum (closed circles) or L. donovani serum (open circles). n = 5/group. No significant differences between groups were observed at any time point (P > 0.05; Student’s t-test).

Figure 3

Depletion of red pulp macrophages reduces S. pneumoniae clearance in L. donovani-infected mice. A: Naive mice or mice at day 42 after L. donovani infection were treated i.v. with clodronate liposomes 24 hours before S. pneumoniae challenge. The graph shows mean ± SD torso bioluminescence after an S. pneumoniae challenge in untreated naive mice (closed circles) or naive mice treated with clodronate liposomes (closed squares) and in L. donovani-infected mice (open circles) or L. donovani-infected mice treated with clodronate liposomes (open squares). n = 5/group. Numbers indicate P values against the relevant control group at the selected time point. ⁺One survivor only at 4 hours. *All mice succumbed to infection before the 18-hour time point. B: Cryosections of spleen from naive control and clodronate liposome-treated mice (top panels) or from L. donovani-infected control and clodronate liposome-treated mice stained for expression of CD68 (green), CD169 (red), and SIGNR1 (blue). Original magnification, ×200. C: Splenic populations from naive mice (left panels) or L. donovani-infected mice (right panels) stained with antibody against SIGNR1 or with isotype control antibody. Red pulp macrophages (RPM) were defined as CD68^HICD169⁻FSC^HISSC^HI cells. DCs were CD11c^HIF4/80⁻ cells. D: Putative MZMs in naive spleens were SIGNR1^HI cells (E) (R1-gated) within the CD68⁻CD169⁻ FSC^HISSC^HI population.

Figure 4

Depletion of specific phagocyte populations does not impair early S. pneumoniae clearance in L. donovani-infected mice. Naive mice or mice at day 42 after L. donovani infection were treated i.v. with depleting antibodies against defined phagocytic cell populations 24 hours before S. pneumoniae challenge. A: Cryosections of spleen from L. donovani-infected control and anti-Ly6C/G- and anti-Ly6G-treated mice stained for expression of 7/4 antigen (green) and CD68 (red). Original magnification, ×200. B: Cryosections of spleen from naive B6 mice treated 48 hours previously with DTx (left) and of spleen from L. donovani-infected B6.CD11cDTR control (center), and DTx-treated mice (right) stained for expression of CD169 (green) and CD11c (red). Original magnification, ×200. C–E: Graphs show mean ± SD torso bioluminescence for control and antibody/DTx-treated groups after S. pneumoniae challenge. C: Naive untreated control mice (open squares) or naive mice pretreated with isotype control (closed squares) or anti-Ly6C/G (anti-neutrophil/monocyte; closed circles). D: L. donovani-infected mice pretreated with isotype control antibody (closed squares), with anti-Ly6C/G (closed circles), or with anti-Ly6C (anti-neutrophil; open circles). E: L. donovani-infected CD11cDTR mice (closed squares), and L. donovani-infected CD11cDTR mice pretreated with DTx (open squares). mAb, monoclonal antibody.

Figure 5

Splenic distribution of S. pneumoniae. A and B: The localization of fluorescein isothiocyanate-dextran (left) and PKH26-labeled S. pneumoniae (center) are shown in spleens of naive (A) and L. donovani-infected (B) mice 30 minutes after i.v. administration. Composite images (right) are also shown. Original magnification, ×400. Insets: arrows indicate colocalized dextran and S. pneumoniae, and circles indicate S. pneumoniae within dextran-negative populations.