Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin

Age-matched properdin-deficient and wildtype mice (C57Bl/6 background) were taken from the colony generated and maintained at the University of Leicester (8–12 weeks old) [6]. Experiments were performed in accordance with UK Home Office regulations and institutional ethical approval.

Streptococcus pneumoniae serotype 2, strain D39, was passaged in MF1 mice as standard procedure to maintain virulence. For infection, mice were anaesthetised with 2.5 % (v/v) fluothane (AstraZeneca, Macclesfield, UK) over oxygen (1.5–2 l/min), and pneumococcal suspension (1 × 10⁶ CFU in PBS, 50 μl) was administered intranasally (i.n. model). Intravenous infections were administered via the tail vein using the same dose (100 μl i.v. model). Dose concentration was confirmed by plating on blood agar. Mice were monitored for signs of illness throughout the 7 days of the experiment and culled before reaching the endpoint of severely lethargic. This timepoint was recorded as the survival time. Mice alive at 7 days post-infection were deemed to be survivors. Vaccination with Pneumovax was performed by single intraperitoneal (i.p.) injection as described [23].

Lung homogenates from control and infected mice were analysed by Western blot using HRP-conjugated goat antimouse C3 (ICN pharmaceuticals Inc.), and the density of bands corresponding to the C3 activation products was measured.

Bloods were collected under terminal anaesthesia by cardiac puncture. Sera from infected and control mice (S. pneumoniae model) were stored in aliquots at –80 °C until required. Sandwich ELISAs were used to determine IgM (Bethyl Laboratories, Universal Biologicals Ltd., Cambridge UK) and total C3 levels (Immunology Consultants Laboratory Inc, Immune Systems Ltd., Paignton UK). To determine antipolysaccharide 2 IgM antibodies, plates were coated with purified serotype 2 pneumococcal polysaccharide (ATCC, Middlesex, UK) [24] and serum was added (1:300 dilution). Importantly, serum samples were pre-absorbed (37 °C, 1 h) with purified pneumococcal cell wall polysaccharide (CWPS Statens Serum Institute, Denmark; 5 µg/ml) to neutralise antibodies against cell wall polysaccharide before antibodies to PPS were detected using the antigen capture ELISA. C4c levels were determined using a previously described method [25].

A bioassay was performed using actinomycin sensitised L929 indicator cells, and levels were expressed in relation to a TNF-α standard (Peprotech EC Ltd., London).

Listeria monocytogenes serovar 1/2a, strain EGD-e (obtained from American Type Culture Collection) was passaged in C57Bl/6 mice. 1 × 10⁶ CFU in PBS were injected in a tail vein (100 μl). The number of viable, inoculated bacteria was determined by colony counts. The mice were monitored for signs of illness and culled before reaching a lethargic state. Mice alive at 96 h were considered to have survived the infection.

Mice were culled, and livers were homogenised in 10 ml PBS or dH₂O as above. The numbers of CFU/g of liver tissue were determined by plating serial 10-fold dilutions of homogenates on BHI-agar plate (37 °C, 24 h). The colonies showed typical morphology (small, round colonies with a bluish-green sheen when viewed by obliquely transmitted light).

Bone marrow cells were prepared using standard methodology by flushing tibias and femurs with PBS using a 0.45 mm syringe. The supernatant, after sedimentation of debris, was centrifuged, and erythrocytes were lysed using a hypotonic solution. After neutralisation, cells were seeded at 2 × 10⁶/ml in the presence of IL-4 and GM-CSF (each 10 ng/ml; Peprotech). After 7 days, non-adherent cells were separately collected from those cells, which became adherent under these conditions. Both populations were used separately for in vitro infection in antibiotic free complete culture medium (RPMI 1640, 10 % (v/v) FCS) with unpassaged, washed L. monocytogenes (at a multiplicity of infection, or MOI, of 0.2) in mid exponential growth phase, following the method described in a previous study [26].

cDNA was transcribed from 1 μg total RNA prepared from cells or tissues using First Strand synthesis kit (Invitrogen). Gene-specific amplification using SensiMix SYBR kit (Bioline Reagents Ltd., London) was analysed with Rotor-Gene 6000 (Corbett Life Science) and expressed in relation to the housekeeping gene using the ΔΔCT method. Primer sequences for this were mouse FcgR2b: 5′-CTGAGGCTGAGAATACGATC-3′ and 5′-GTGGATCGATAGCAGAAGAG-3′; for mouse FcgR4: 5′-GTGACCCTCAGATGCCAAGGC-3′ and 5′-TGGATGGAGACCCTGGATCGC-3′, for mouse β-actin: 5′-GTGGGCCGCTCTAGGCACCAA-3′ and 5′-CTCTTTGATGTCACGCACGATTTC-3′, for mouse IL-17A 5′-GGCTGACCCCTAAGAAACC-3′ and 5′-CTGAAAATCAATAGCACGAAC-3′. Annealing temperatures were 60, 68, 55 and 53 °C, respectively.

Oligonucleotides used for standard amplification of murine transcripts for TLR2, CD11b and C3 (annealing temperature 55 °C) were 5′-GGCCAGGTTCCAGTTTTCAC-3′ and 5′-GGAACAACGAAGCATCTGGG-3′; 5′-GATGGTGTCGAGCTCTCTGCG-3′ and 5′-TTGTCTCAACTGTGATGGAGCA-3′; 5′-GAATACGTGCTGCCCAGTTT-3′ and 5′-TGAGTGACCACCAGCACTTT-3′, respectively.

To quantify the phagocytic uptake of S. pneumoniae D39 by splenic macrophages, extracellular fluorescence was quenched with 0.2 (w/v) % trypanblue prior to flow cytometry. To prepare the labelled bacteria, S. pneumoniae D39 were grown in broth (stationary, 37 °C), and 10⁹ bacteria/ml were resuspended in 0.1 mg/ml FITC (Sigma-Aldrich, 0.1 M NaHCO₃ pH 9.0). After 60 min at room temperature, bacteria were spun and washed in PBS until the supernatant no longer was yellow. Splenic cell suspensions were prepared from a pool each of two properdin-deficient and wildtype mice, and adherent cells were incubated in the presence of genotype-matched serum with labelled S. pneumoniae for 1 h prior to analysis.

Events were acquired using Becton–Dickinson FACS Scan and CellQuest pro software.

Survival curves were compared using the log rank test. Other data were analysed using t tests. The difference in sequentially measured CFU in blood from infected mice was analysed by Mann–Whitney U test.

An intact complement system is necessary in the immune response to encapsulated microorganisms [27]. To investigate the response of properdin-deficient mice to capsular polysaccharides in comparison with wildtype mice, mice were immunised i.p. with Pneumovax, a 23-valent pneumococcal polysaccharide vaccine. Natural antipolysaccharide IgM antibodies were detectable before vaccination. At the end of the four-week experimental period, both genotypes had raised an IgM antiserotype 2 capsular polysaccharide response (Fig. 1a). To determine whether they were protective, immunised mice were challenged with serotype 2 S. pneumoniae by i.n. inoculation, which normally proceeds from bronchopneumonia to sepsis within 24–48 h. While both genotypes immunised with Pneumovax were protected in the challenge infection (Fig. 1b), localising pneumococci to alveolar macrophages (Fig. 1c), the survival of the non-immunised mice infected in parallel was significantly different (Fig. 1b): while wildtype mice succumbed rapidly, with 2/5 alive at the experimental endpoint of 7 days, 4/5 properdin-deficient mice remained alive. To confirm this finding, additional groups of naïve mice were infected with an independently prepared stock of passaged S. pneumoniae, and again, properdin-deficient mice showed significantly greater survival compared to wildtype mice (8/10 vs 4/10) (Fig. S1A). They also had significantly milder disease at 24 h (Fig. S1B). Individual mice were sampled at various timepoints p.i. and viable pneumococcal counts in lungs and blood determined; while wildtype mice appeared to control bacterial numbers in lungs at 48 h compared to 24 h, as expected for mice of this genetic background, properdin-deficient mice failed to do so (Fig. 2a). In fact, properdin-deficient mice had the highest bacterial count in blood at 48 h (Fig. 2b), from where bacteria may reseed the lungs (personal communication, A. Kadioglu). The differential count of neutrophils in lung homogenates prepared from wildtype and properdin-deficient mice at all timepoints was not significantly different from each other though relative numbers peaked at 24 h. At this timepoint, sublocalisation of neutrophils was investigated histologically (parenchymal vs tethered). There was no difference between the infected genotypes, though parenchymal neutrophils expectedly were elevated compared to controls. The percentage of capillary neutrophils tethered to the endothelium was also comparable between the infected genotypes (Suppl. Table 1). Histological examination of lung sections at this timepoint showed regional leukocytic infiltrations, hyperaemia and thickening of interalveolar spaces, all typical signs of pneumonia. Serum C3 levels and C3 activation fragments in the lung were increased over baseline for both genotypes and were higher for properdin-deficient than for wildtype mice at 48 h (supplementary Table 2), when the viable count was highest in blood and lungs of properdin-deficient mice.

Previously, activation of the classical pathway of complement was shown to be important in murine survival of S. pneumoniae [10]. Wildtype and properdin-deficient mice showed a significant decrease in circulating IgM levels as early as 24 h (Table 1), likely to be due to binding to S. pneumoniae and sequestration from the fluid phase. C4c, a product of complement activation, was significantly increased in the sera from infected wildtype, but not in the sera from properdin-deficient mice compared to their respective, comparable, baselines (Table 1), consistent with greater complement activity in properdin-sufficient wildtype mice. In keeping with this, association of C3 to S. pneumoniae in vitro via the classical pathway was significantly higher after incubation with sera from wildtype than from properdin-deficient mice (Fig. 3a). To compare opsonophagocytosis in the two genotypes, splenocytes were isolated from wildtype and properdin-deficient mice and incubated for 1 h with S.pneumoniae previously incubated with a pool of sera from either wildtype or properdin-deficient mice (Fig. 3b, c). S. pneumoniae opsonised with serum from wildtype associated significantly more with splenocytes prepared from wildtype mice compared to the matched properdin-deficient experimental sample. As a consequence, splenocytes from wildtype mice released significantly more TNF-α compared to those from properdin-deficient mice after infection with S. pneumoniae. During intravenous infection with S. pneumoniae, the rate of bacterial growth from 24 to 72 h was the same in the two genotypes, but the bacterial burden was significantly higher in properdin-deficient mice (Fig. 3d).

Taken together, the experiments thus far revealed that in the presence of the alternative pathway amplification loop, anticapsular antibodies activate the classical pathway of complement and enhance phagocytosis and proinflammatory cytokine release, the extent of which was detrimental to the wildtype host, while in the absence of properdin, clinical signs of pneumococcal septicaemia were less severe, even though the viable counts recoverable from blood were higher than in wildtype mice.

Following a report that survival of pneumococcal sepsis was improved in the absence of the inhibitory FcγR2b receptor [29]—and others dealing with the crosstalk between complement and FcγR expression [30, 31]—we investigated FcγR2b expression in properdin-deficient and wildtype mice. The mRNA expression of FcγR2b in spleen was reduced in properdin-deficient mice (Fig. S2A). After in vitro infection of splenocytes with S. pneumoniae, however, cells from wildtype mice decrease their expression while properdin-deficient cells show more abundant expression, supporting the pro-inflammatory phenotype in wildtype mice shown in Fig. 3c. The activating receptor FcγR4 (functionally antagonistic to FcγR2b), in contrast, is inversely expressed in wildtype and properdin-deficient spleens (Fig S2B). To unequivocally demonstrate a difference in expression of FcγR2b between wildtype and properdin-deficient mice, splenocytes from age- and sex-matched mice were analysed by flow cytometry for expression of FcγR2b. Because the available monoclonal antibodies recognise both FcγR2b and FcγR3, analysis was restricted to B lymphocytes, which only express the inhibitory FcγR2b receptor. Again, properdin-deficient mice had reduced expression of FcγR2b (Fig S2C).

Because infection with S. pneumoniae unexpectedly revealed a cellular phenotype of properdin deficiency (low FcγR2b expression, decreased phagocytosis), mice were next analysed in in vitro and in vivo models using an intracellular pathogen, L. monocytogenes. As for S. pneumoniae, mouse serum was not bactericidal for L. monocytogenes (data not shown), which is consistent with the literature [11, 32].

Bone marrow-derived cells were differentiated in the presence of IL-4 and GM-CSF, yielding an adherent, macrophage-like and non-adherent, dendritic cell-like population [26]. Cells were infected with live L. monocytogenes. We found that both cell populations increased their mRNA expression for C3, the integrin CD11b and TLR2 (data not shown). Infected macrophage-like cells from wildtype released marginally more TNF-α than those from properdin-deficient mice when infected with live, not heat killed, Listeria (data not shown). Remarkably, however, the dendritic cell-enriched population from properdin-deficient mice had significantly lower intracellular Listeria compared with those from wildtype mice, while there was no difference in the intracellular burden for the adherent cell population from the two genotypes (Fig. 4a). Release of IFN-γ from infected cells from properdin-deficient mice was lower compared to wildtype mice (Fig. 4b). Nitric oxide release was induced by infection, to the greatest extent in the dendritic cell-like population from wildtype mice (Fig. 4c). Electron microscopy was used to determine the subcellular localisation of L. monocytogenes. Consistent with the greater viable counts determined in the dendritic cell-like population from wildtype mice compared with those from properdin-deficient mice, there were more Listeria present in the vacuoles and cytosol of wildtype dendritic cell-like cells (Fig. 5a–f). Macrophage-like cells, however, did not follow this pattern. In contrast, there was a greater number of Listeria in the cytosol at 24 h than in vacuoles of cells from wildtype, not properdin-deficient mice, possibly indicative of lysosomal escape of L. monocytogenes (Fig. 5g–l).

Listeriolysin was detected in supernatants of infected splenocytes of wildtype and properdin-deficient mice by Western blot (data not shown). Therefore, to preserve the viability of splenocytes for flow cytometry, heat-killed L. monocytogenes were used. Compared to the wildtype, the dendritic cell-enriched population of properdin-deficient bone marrow was impaired in the upregulation of CD40, an accessory molecule needed to prolong contact between antigen-presenting and T cells (Fig. 6). Surface expressions of MHCII, CD80 and CD86 were increased after infection, indicating maturation of cells, but were not appreciably different between the genotypes (Fig. S3). A selective impairment of CD40 upregulation in dendritic cells isolated from properdin-deficient mice—contrasting with MHCII, CD80 and CD86—had previously been observed when maturing bone marrow-derived dendritic cells from both genotypes with 1 μg/ml LPS E.coli 0111:B4 for 48 h (N. Salehen, PhD thesis).

An in vivo model of murine listeriosis reproducibly showed that complement properdin contributed significantly to host survival (Fig. 7a) though the numbers of bacteria retrieved from infected livers were not different between the two genotypes (Fig. 7b). After 2 days, 7/9 wildtype mice and only 2/9 properdin-deficient mice were alive. The IFNγ levels in serum were significantly elevated in those mice with worse prognosis, i.e. the L. monocytogenes infected properdin-deficient mice (Fig. 7c). Granuloma formation was not impaired in properdin-deficient mice. The titres of antilisterial IgM tested towards immobilised L. monocytogenes were comparable in naïve and infected mice (data not shown).

The host normally responds to infection with L. monocytogenes with an increase in IL-17 [33], which aids in the polarisation of M1 macrophages [34]. The level of complement activity reportedly affects IL-17 production by T cells and macrophages [35]. When investigating the expression levels in control properdin-deficient and wildtype mice, we found significantly impaired basal expression of IL-17A mRNA in properdin-deficient mice (Fig. 7d). After infection with L. monocytogenes, mice of identical severity culled at 29 h were analysed for their splenic IL-17A mRNA expression. Properdin-deficient mice were again reduced in their expression by threefold though the overall abundance of mRNA was higher than unstimulated controls (Fig. 7d). In support of this observation, the relative reduction in inflammatory splenic IL-17A mRNA expression in the absence of properdin was also found when analysing mice infected with a lower dose (5 × 10⁵ L. monocytogenes) and survival to 49 h (n = 4 WT, n = 1 KO) (data not shown).

Previously, we have shown that properdin-deficient mice generated by site-specific targeting [6] were impaired in their alternative pathway activity [6, 7], which led to impaired amplification of the classical pathway of complement activation [36]. Lower levels of C5a in properdin-deficient mice [7, 36] concur with reduced expression of FcγR2b on phagocytic cells, resulting in cells impaired in their ability to phagocytose-encapsulated microorganisms such as S. pneumoniae [29].

Mice held in specific pathogen-free environments express natural IgM antibodies reactive with capsular polysaccharides, including those specific for S. pneumoniae serotype 2 (Fig. 1a). The anticapsular polysaccharide 2 IgM antibody response after immunisation with the polysaccharide pneumococcal vaccine was comparable between properdin-deficient and wildtype mice. Their total IgM levels were not elevated compared to unvaccinated mice. Mice deficient of C3 or CR2 (the B cell complement receptor) were also able to increase their serotype 2 capsular polysaccharide reactive IgM after immunisation [28], negating concerns that a decrease in C3 fragments would increase the threshold for antibody production [reviewed in [45]]. However, it is worth noting that the immunisations were performed by the i.p. route, stimulating primarily peritoneal B-1-cells [46]. The protective effect of vaccination was tested by i.n. infection with S. pneumoniae D39 (serotype 2) and verified for wildtype mice. Properdin-deficient mice, however, reproducibly demonstrated a survival advantage in this model—which was inherent—even though the bacterial load in lungs and systemic seeding into the blood at 48 h post-infection was higher. Their clinical severity scores were significantly better than those obtained for infected wildtype mice.

The increase in serum C3 as an acute phase marker in the inflammatory response showed no difference between the two genotypes. Pulmonary C3 fragments, however, were higher for the infected properdin-deficient group though numbers and location of neutrophils (parenchymal, vascular) were comparable to the infected wildtype group. Given that the bacterial viable count was elevated in lungs of properdin-deficient mice, it is possible that S. pneumoniae themselves exerted proteolytic activity against C3, as shown by others [47].

Against these novel findings and given that the response towards zymosan, being rich in protein–carbohydrate complexes, also requires natural antibodies [48], the survival advantage of properdin-deficient mice in our zymosan shock model [7] is likely to have been due to a qualitatively different immune response as outlined above, rather than a beneficially impaired inflammatory ability in the absence of properdin.

To understand the role of properdin in cell-mediated responses during infection with L. monocytogenes, we investigated phenotypic changes in differentiated bone marrow cells from properdin-deficient and wildtype mice. When infected with the same MOI, the dendritic cell-like population prepared from properdin-deficient mice had reduced numbers of intracellular bacteria compared to that from wildtype mice. As a consequence, these cells were also impaired in their antigenic maturation compared to those from wildtype mice after infection with live L. monocytogenes (lower CD40 expression on CD11c⁺ cells). They did not differ in the expression and maturation-induced upregulation of MHCII, CD80 and CD86, thereby expanding from other work, which concluded that C3 was not necessary for maturation of dendritic cells in L. monocytogenes infection [39]. CD40, however, was not assessed in these in vitro analyses of dendritic cells from C3 deficient mice [39]. The substantial impairment of CD40 upregulation in properdin-deficient CD11c⁺ cells implies less efficient interactions with cognate T cells. This observation is corroborated by the fact that properdin-deficient mice showed little cytosolic presence of Listeria in their dendritic cell-like population, consistent with impaired cell activation and secretion of IFNγ: cytosolic entry of virulent Listeria is required to increase expression of co-stimulatory molecules and to generate an appropriate T-cell response [49, 50].

M2-skewed myeloid cells express lower CD40, lower IFNγ, TNF-γ and nitrites than M1-polarised cells [51]; this coincides with lower production of IL-17 [52]. Using these parameters, our study gives consistent evidence that cells isolated from properdin-deficient mice retain in culture a spectrum of activity compatible with M2 polarisation.

IFNγ is significantly increased in serum of infected properdin-deficient mice compared to wildtype mice, while both readings were elevated compared to serum from naïve mice. Given that the two genotypes exhibited equal numbers of viable counts in liver homogenates during listeriosis, the increase in IFNγ was likely to be the attempt of properdin-deficient mice to counteract M2-type activity, which is detrimental in listeriosis [53].

Taken together, the juxtaposed studies demonstrate that in the attempt to understand the opposing clinical outcomes of the infection models used, a role of properdin deficiency in M2 skewing has been uncovered (Fig. 8). Some determinants of outcome for properdin-deficient mice described for the listeriosis model are likely to have a role in the pneumococcal model via their contribution to M2 skewing, such as IL-17 [54]. Conversely, in wildtype mice, we now conclude that properdin plays a role in maintaining the M1 phenotype favoured by the genetic background of C57Bl/6. Given that there is accumulating literature on a role for complement in cellular immune responses, it follows that the findings presented herein pose relevant considerations in the design of complement targeting biomolecules in general and of properdin blockers in particular.

Finally, though the studies presented herein have advanced the appreciation of the crosstalking role of complement in vivo, ex vivo and in vitro, one relevant contributor to the host response could not be analysed; to profile the virulence of S. pneumoniae and L. monocytogenes in blood and tissues, an expansion in bacterial growth medium was needed, which provokes another environmental adjustment of the transcriptomes. It is important to recognise that because the pathogens are exposed to selection pressures, which are modulated in the gene-targeted experimental animal, the pathogens are likely to be phenotypically different between the mouse genotypes and therefore are likely to differentially co-determine the infection outcomes in these models. The extent of this contribution is currently under investigation.