PD-L1 expression in schistosomal granuloma: an immunohistochemical study in urinary bladder schistosomiasis

Abstract

Aims

Schistosomiasis remains endemic in various parts of the world, and insights into pathogen immunobiology are mainly based on experimental models, while studies on human tissues are limited.

Methods

We explored the role of immune checkpoint pathway by evaluating the immunohistochemical expression of programmed death-ligand 1 (PD-L1) in a retrospective cohort of patients with bilharzial cystitis. Inflammation severity by conventional histology and staining intensity by immunohistochemistry were assigned three-tier scores (0/1+/2+), and a cut-off for staining percentage was set at 5%.

Results

38 biopsies from 31 patients were considered adequate for evaluation, and positive staining was detected in 80.6% of patients (34 biopsies). High expressors (22.6%) showed strong positive membranous staining (score 2+) with high staining density (more than 5% of inflammatory cells). Low expressors (58.1%) showed mild/moderate staining (score 1+) predominantly in less than 5% of the cells (91.6%) or expressed restricted cytoplasmic staining (6/31). All high expressors showed severe inflammation (score 2+) (p<0.001), and viable ova were only observed in these cases. Calcified ova were associated with mild/moderate inflammation or absent/minimal inflammation, correlating with low expressors or non-expressors (19.4%), respectively.

Conclusion

Schistosomal granuloma exhibits upregulated PD-L1 expression proportional to inflammation severity and pathogen viability, highlighting a critical immune checkpoint engagement in disease pathology.

WHAT IS ALREADY KNOWN ON THIS TOPIC

• Schistosomiasis affects nearly half a billion individuals worldwide despite global efforts for control, and a comprehensive understanding of immunobiology and mechanisms underlying morbidity remains elusive.

WHAT THIS STUDY ADDS

• Programmed death-ligand 1 is overexpressed in peri-oval granuloma with strong correlation with inflammation severity, suggesting an immune-evasive mechanism at certain phases of infection.

HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

• Evidence of immune checkpoint pathway activation in patients with schistosomal cystitis paves the way for enhanced characterisation of host-pathogen interactions in non-experimental settings.

Introduction

Schistosomiasis is a grave public health problem; according to global estimates, at least 230 million people are actively infected and nearly an equal number endure post-infection sequelae.¹ The causative organism is a water-borne trematode of the Schistosoma genus, and human infections are mainly caused by three species: Schistosoma haematobium, Schistosoma mansoni and Schistosoma japonicum.²

After exposure to contaminated water, the cercariae (larval form of schistosoma) penetrate the skin, then travel through the circulation, where they mature into adult worms that selectively lodge in the perivesical (S. haematobium) or mesenteric venous plexuses (S. mansoni and S. japonicum).³ The intravascular separate-sexed worms have an exceptional longevity reaching up to 30 years inside their mammalian host, during which the female adult lays hundreds to thousands of eggs daily that are passed through the bladder or intestinal lumina.⁴

Tissue-impacted ova are the predominant cause of morbidity in schistosomiasis. In the urinary bladder, the accompanying inflammatory and fibrotic reactions cause hydroureter and hydronephrosis with subsequent renal dysfunction or bladder squamous cell carcinoma. On the other hand, embolisation of S. mansoni and S. japonicum ova to the liver vasculature is complicated by ascites or potentially fatal oesophageal variceal bleeding secondary to portal hypertension with preceding hepatic fibrosis.⁵,⁷

The immunopathology of schistosomiasis is quite complex, and diverse immune responses are triggered and directed towards various life stages of the parasite.⁸ It is intriguing how such organisms can thrive despite vigorous immune reactions, and it is hypothesised that micro-organisms can escape, dampen or even use defensive immunity to their benefit, maintaining survival within the host.⁹

Studies in animal models have demonstrated that adult worms induce CD4 and CD8-positive T-cell anergy via selective upregulation of programmed death-ligand 1 (PD-L1) on macrophages.¹⁰ Such an interesting observation raises the question of whether schistosomiasis might be using the same immune evasion mechanism of the programmed cell death protein-1 (PD-1)/PD-L1 pathway that was found to be frequently employed in various cancers.¹¹

In order to investigate the potential role of the immune checkpoint pathway in human schistosomiasis, we studied the immunohistochemical (IHC) expression of PD-L1 in patients with S. haematobium infection of the urinary bladder.

Materials and methods

Material acquisition

Bilharzial infection is uncommon in the USA; therefore, the material was shared by a collaborating healthcare institution in Egypt, where bilharziasis is still endemic. The electronic pathology database was searched during the period 2020–2021 for the keywords “Bilharzial Cystitis”, and the diagnostic pathology reports were reviewed, patients that had associated pathology, such as urothelial carcinoma were excluded. The cohort included 48 biopsies from 37 patients; 46 biopsies were obtained from various areas within the urinary bladder, while the remaining two biopsies were from the prostatic urethra. Diagnostic tissue blocks for all patients were retrieved from the archives and anonymised.

IHC staining

The IHC staining was carried out using PD-L1 (clone E1L3N) rabbit monoclonal antibody (catalogue #13684) from Cell Signaling Technologies (Danvers, Massachusetts, USA) according to the manufacturer’s specifications, and as previously outlined.^{12 13} In brief, the antibody was optimised prior to tissue staining, and each run included control testing to ensure staining reliability. Human tonsil tissue was used as a control, E1L3N-stained slides were a positive control, and a matched isotype was a negative control. The formalin-fixed, paraffin-embedded tissue blocks were cut at 5 µm in thickness and deparaffinised by BOND Dewax Solution, 100% Alcohol and BOND Wash Solution. Antigen retrieval was performed with the BOND Epitope Retrieval ER2 Solution, followed by incubation. The Leica Bond RX stainer (Leica) was used for staining, and the antibody was diluted to 1:400 in Background Reducing Diluent (Dako) before incubation. The Leica Refine Detection System included hydrogen peroxidase block, post-primary and polymer reagent, DAB and haematoxylin. Slides were rinsed and dehydrated in increasing concentrations of ethyl alcohol and cleared in xylene, then mounted with coverslips.

Histological interpretation

Assessment of morphology and IHC staining was carried out by two pathologists (JCC and REE). All cases were initially examined in the routine H&E stain to confirm the presence of bilharzial ova as a marker of schistosomal infection, and the status of these ova, whether recent and viable or old and calcified was noted. Biopsies with significant processing artefact or insufficient residual tissue precluding adequate assessment were excluded (n=8). Following initial screening and evaluation, the degree of inflammation was scored semi-quantitatively in a three-tier fashion as (1: absent to minimal, 2: mild to moderate and 3: marked). Surface epithelial metaplastic or proliferative changes were also documented.

Evaluation and scoring of IHC staining

Interpretation of the IHC stain included assessment of staining presence, intensity, percentage and pattern. Cases were labelled as ‘positive’ if staining of any intensity was noted in the granuloma or aggregates of inflammatory cells surrounding and related to the schistosoma ova. Staining scoring was assigned as: score 0 (indicating absence of staining), score 1+ (mild to moderate staining that was more evident with higher magnification) and score 2+ (indicating strong staining that was readily visible even on low magnification). In instances where variable staining intensities were noted in the same biopsy, the final score was based on the highest intensity detected. Similarly, for patients with multiple biopsies, the highest scoring value was used. A reference point for this scoring method was achieved through comparison with the antibody positive controls (tonsillar tissue in this study), where strong expression (equivalent to score 2+ in our cases) was present in the tonsil crypt epithelium, while weak to moderate staining (equivalent to our proposed score 1+) was noted in the follicular macrophages of the germinal centre.

Staining percentage referred to the number of positive cells out of the total inflammatory infiltrate surrounding and adjacent to ova within each case. To simplify the evaluation, a 5% cut point was set, and cases were labelled as having either more or less than 5% staining in a binary fashion. The pattern of expression, whether membranous or cytoplasmic or both, was documented for all cases. Demonstration of positive internal control was achieved in all biopsies (including score 0 cases). If marked surface ulceration was detected in a biopsy, evaluation of staining was directed to the deeper portions of the biopsy away from the surface.

Statistical analysis

Descriptive analysis was carried out for all variables, nominal and ordinal data were reported as absolute number and proportions (%). The Spearman correlation test was used to test the relation between inflammation, staining intensity and percentage. P values <0.05 were considered statistically significant. Statistical analysis was done using V.28.0 of IBM SPSS (IBM SPSS Statistics for Windows, Armonk, New York: IBM Corp).

Results

Morphology

Biopsies from 31 patients were considered adequate for evaluation. The degree of inflammation was variable between cases, 10 biopsies from 7 patients (22.6%) showed marked inflammatory reaction directly surrounding the ova and in the adjacent tissue (figure 1), 16 biopsies from 13 patients (41.9%) had mild to moderate inflammation, whereas minimal or absent inflammation was observed in 11 cases (35.5%, 14 biopsies) (table 1). Viable bilharzial ova were only noted in cases with severe inflammation (22.6%), while old and calcified ova were seen in all the study cases, including cases with marked inflammation. Epithelial metaplastic changes included squamous metaplasia in four cases (12.9%), extensive glandular metaplasia was observed in one case (3.2%) and marked proliferative changes in the form of abundant von Brunn nests were also observed in one case.

Figure 1. Morphology: low and intermediate power view (panels a and b, respectively) showing H&E-stained sections with dense tissue infiltration by viable ova surrounded by extensive inflammatory reaction (10× and 20×). High power view of schistosomal ova (arrows) surrounded by multinucleated giant cells (panel c) or by numerous eosinophils (panel d), 40×. Extensive surface squamous metaplasia with few calcified ova (arrows) in the submucosa (panel e, 4×). One case showed the undulating pigmented silhouette of an adult worm embedded in the bladder wall (arrowheads), while the inset shows another field where a portion of the adult worm is seen in a vesical venule, panel f, 10×.

Table 1. Summary of morphological and immunohistochemical features.

Morphological/immunohistochemical feature	Scoring	Number and percentage (N=31)
Inflammation intensity	Score 1 (absent to minimal)	11 (35.5%)
	Score 2 (mild to moderate)	13 (41.9%)
	Score 3 (marked)	7 (22.6%)
PD-L1 staining intensity	Score 0 (absent)	6 (19.4%)
	Score 1+ (mild to moderate)	18 (58.1%)
	Score 2+ (strong)	7 (22.6%)
PD-L1 staining extent	More than 5% of inflammatory cells	8 (25.8%)
	Less than 5% of inflammatory cells	17 (54.8%)
PD-L1 staining pattern	Membranous	19 (61.3%)
	Cytoplasmic	25 (80.6%)

PD-L1, programmed death-ligand 1.

Immunohistochemistry results

Biopsies from 25 patients (80.6%, 34 biopsies) showed PD-L1 expression in the granuloma surrounding parasitic ova, and staining was predominantly observed in the macrophages and multinucleated cells, and to a lesser extent, in lymphocytes. Seven cases (22.6%) showed strong membranous PD-L1 expression (intensity score 2+) (figure 2, panels b–e). 18 cases (24 biopsies) representing 58.1% showed mild to moderate staining (score 1+) (figure 2, panel f), while 6 cases (19.4%) showed absence of staining. Membranous expression was noted in 19 cases (61.3%), and all these cases also showed at least focal cytoplasmic staining; however, restricted cytoplasmic staining was noted in 6 cases (19.4%). Staining in more than 5% of inflammatory cells was noted in 8 cases (25.8%), and the remaining 17 positive cases showed staining in less than 5% (54.8%) (table 1).

Figure 2. Immunohistochemistry: Programmed death-ligand 1 (PD-L1) expression was readily identifiable at variable intensities even at low power magnification (panel a, 4×). Panel b demonstrates strong expression in the inflammatory cells surrounding the bilharzial ova (arrowheads) at intermediate magnification (20×), while panel c shows higher magnification (40×) of a densely cellular focus with extensive PD-L1 staining. Panel d showing high-power view (40×) of a less cellular focus where the membranous staining pattern could be appreciated surrounding the parasitic ova (arrow). PD-L1 stain highlighting the cell borders of multinucleated giant cells (arrows) and surrounding cells (panel e, 40×). Panel f showing section from a case with score 1+ intensity where mild to moderate membranous staining was noted in inflammatory cells, 40×.

Based on the staining intensity, extent and pattern, cases were stratified into two groups: ‘high expressors’ and ‘low expressors’. The first group included cases with score 2+ intensity, that was observed in more than 5% of inflammatory cells and was predominantly membranous (seven cases, 22.6%). The ‘low expressors’ (18/31, 58.1%) included cases with score 1+ staining, where expression was detected in less than 5% of cells in all except one case. Cases with restricted cytoplasmic staining (n=6) were included in the second group.

Correlation of morphology and immunohistochemistry results

The degree of inflammation seemed to correlate with staining intensity (p<0.001), and cases with marked inflammatory response all showed score 2+ staining; on the other hand, all cases with mild to moderate inflammation all showed weaker staining (score 1+). The degree of inflammation also correlated with the percentage of PD-L1 positive cells (p<0.001), and markedly inflamed cases had higher counts of positive cells (more than 5%), while all except one case with mild/moderate inflammation had lower positive cell counts (less than 5%).

Discussion

Bilharziasis is an ancient illness; illustrations on Egyptian medical papyri describe symptoms of a disease resembling bilharziasis, and schistosomal ova were recovered from the kidneys of Egyptian mummies dating as early as the 20th dynasty.^{14 15} Over a century has passed since the initial discovery of schistosomal worms in 1851 by Theodor Bilharz (German physician who at that time was chief of surgery at Kasr-El-Ainy Hospital, Cairo, Egypt) during a postmortem examination¹⁶; however, despite all efforts to control the disease, the annual mortality rate reaches 200 000, and the WHO estimates that more than 700 000 people are at risk of infection.^{17 18}

To the best of our knowledge, this is the first study conducted on human tissue aiming to assess PD-L1 IHC expression in Schistosoma-associated inflammation in a non-cancer setting. Some studies have used immunohistochemistry to study PD-L1 expression in tumour and immune cells of Schistosoma-induced cancers; however, PD-L1 status in the peri-parasitic inflammatory milieu was not investigated, and associations between infection and upregulation of tumorous PD-L1 could not be established.¹⁹,²¹

We have demonstrated that the peri-oval granuloma and inflammatory infiltrate can be strongly positive for PD-L1, and that inflammation severity was a clear determinant of degree and extent of PD-L1 expression (p<0.001). Inflammation status was, in turn, primarily dependent on the condition of the ova present; marked inflammation was only witnessed when the ova were viable, while inflammation surrounding calcified ova ranged from virtually absent/minimal to mild/moderate.

Studies on PD-L1 IHC expression in infectious granulomatous lesions from pathological human tissues are scarce. An early contribution to this area came from Inman et al, who studied PD-L1 expression in high-risk urothelial carcinoma patients receiving therapeutic intravesical BCG injection. In their study, PD-L1 was observed in the BCG-granuloma of 11/12 patients who failed BCG treatment with subsequent tumour recurrence. The authors speculated that PD-L1 upregulation could be responsible for harnessing the immune response towards the infection and the tumour, causing its progression.²² Few other studies reported PD-L1 staining in TB-induced granulomas that was mostly observed in the histocytes and multinucleated giant cells.²³,²⁵ Non-infectious granulomatous diseases were also found to express PD-L1, sarcoidosis being the most frequently investigated, in addition to a few reported cases of Crohn’s disease-related granuloma, foreign body granuloma and more recently, granulomatous prostatitis.²⁴²⁶,²⁸

The exact pathophysiology of these findings is still not fully elucidated, and it has been noted that macrophages and multinucleated cells (which result from macrophage fusion) express PD-L1 on interferon gamma (IFNγ) or tumour necrosis factor alpha (TNFα) stimulation.²⁴ In schistosomiasis, these mediators, among many others such as interleukin (IL)-4, IL-5 and IL-13, are involved either in the initial T helper 1 (Th1) or the subsequent T helper 2 (Th2)-biased immune responses that eventually contribute to the granulomatous and fibrotic reactions, accounting for infection-induced morbidity and complications.²⁹,³¹ Previous efforts sought to decipher the role of the immune checkpoint pathway in schistosomiasis, one study has demonstrated that upregulation of PD-L1 resulted in T-cell anergy in murine models, a response that was attributed to the influence of adult worms during acute phases of infection and prior to egg production. It was noted that PD-L1 expression levels declined after 12 weeks of infection, coinciding with the reversal of T-cell anergy.¹⁰ In another study, PD-L2 was reported to be overexpressed in splenic dendritic cells of S. mansoni-infested mice in the early weeks of infection, and such observation was associated with less severe morbidity.³² Similarly, the worms of other parasitic infections, such as Taenia crassiceps, were found to induce PD-L1 and PD-L2 expression on activated macrophages in mice.³³ These tactics were found to be exploited in some bacterial and viral infections as well, including H. pylori, HIV and viral hepatitis.³⁴

Fueled by such observations, attempts at blocking the PD-1/PD-L1 pathway were reported in infectious diseases in the previous literature; however, the outcomes showed substantial variability.³⁵ Immune checkpoint blockers improved viral clearance and reduced viremia in some infections,³⁶,³⁹ unfortunately, restoration of effector T cell function following PD/PD-L blockage resulted in exacerbated tissue damage in several studies on viral and parasitic infections,⁴⁰,⁴² suggesting a possible protective role of this pathway at certain phases of the infectious process. It has been speculated that the PD-1/PD-L1 interplay might be useful in downregulating overactive T cells to help minimise extensive inflammation-induced tissue injury,³⁴ simply to reach a state where both the pathogen and host can ‘survive’.

In schistosomal models, it was reported that the upregulated PD/PD-L axis-associated CD4+ T cell exhaustion contributes to liver fibrosis as the peri-granulomatous CD4+ T cells produce IL-4 stimulating collagen production from murine hepatic stellate cells.⁴³ Surprisingly, the selective augmentation of CD4+ Th2 cell responses following PD-1 signal blockade only caused worsening of liver fibrosis without a significant reduction in egg burden in mice with S. japonicum infection.⁴⁴ A similar expansion of Th2 responses following PD-L1 blockade was observed in a study by Xiao et al, yet their findings indicated that CD4+ response modulation by anti-PD-L1 antibody therapy did not impact hepatic egg burden nor liver pathology (as assessed by hepatic collagen deposition).⁴⁵ Furthermore, Lu et al demonstrated that PD-1 knockout mice exhibited significantly increased T cell counts that—unpredictably—did not contribute to pathogen eradication. Th1 cytokines (IFNγ) were elevated while Th2 response cytokines (IL-4) were suppressed, along with exaggerated regulatory T cell-mediated immunosuppression, fewer eggs and larger liver granulomas compared with controls.⁴⁶

Consequently, it is largely undetermined whether immune checkpoint pathways act as enemies or allies throughout the various stages of infectious diseases’ morbidity, underscoring the need for future clarification, thus, the mere demonstration of PD-1/PD-L1 axis upregulation does not necessarily establish its validity as a therapeutic target.

This dilemma highlights further uncertainties in the evolving field of microbial immunobiology, especially for pathogens like schistosomiasis, where existing knowledge is mainly derived from experimental animal models, but studies on human subjects are far less common. Additionally, the aforementioned efforts did not all conclusively establish the immunosuppressive effects of the ova, which—in humans—are most pathogenic. Therefore, we conducted this study to investigate PD-L1 IHC expression in patients with bilharzial cystitis aiming to outline PD-L1 status in biopsies of human tissues.

Positivity was noted in the majority of patients included (80.6%), and the expression intensity varied between cases and within any given case. Positive staining was generally detectable at low or intermediate magnification, and staining of any intensity was considered positive. We proposed a three-tier scoring system to evaluate all cases (0 vs 1+ vs 2+), which was—in part—inspired by staining variability of the antibody positive control (strong staining in tonsillar crypt epithelium vs weak to moderate staining in follicular macrophages of the germinal centre).

After taking into account staining intensity, pattern and percentage, cases were segregated into high expressors (22.6%) and low expressors (58.1%) groups. Of the whole cohort, only 6/31 cases showed negative staining (non-expressors), which was explained by the scarcity of inflammation that was predominantly formed of lymphocytes with absent macrophages. One case had minimal inflammation but showed a handful of multinucleated giant cells, and in this case, positive membranous staining was detected, highlighting the relation between the presence of macrophages and PD-L1 expression. Less prominent inflammation also explains why staining in the low expressors group was of lower intensity and percentage. Moreover, the degree of inflammation was directly linked to the ova condition (viable vs calcified). All positive cases showed cytoplasmic staining, and in six cases (19.4%), restricted cytoplasmic staining was noted. Such cytoplasmic staining for PD-L1 whether alone or in combination with membranous staining is frequently noted in cancers,⁴⁷ and it was previously documented in the macrophages of TB-induced granuloma.²³ In the cancer-cell context, cytoplasmic staining is usually disregarded, however, for inflammatory cells, it has been emphasised that membrane/cytoplasm staining could be considered positive, especially in lymphocytes where the small cell size impedes accurate distinction between cytoplasmic and membranous staining.⁴⁸ Therefore, cases with restricted cytoplasmic staining in this study were included in the ‘positive expression’ category and placed with the low expressors group.

Hence, the current work not only provides insights into the patterns of PD-L1 expression in Schistosoma-associated inflammation but also proposes a reproducible scoring system for expression assessment. Additionally, we confirm earlier experimental studies that demonstrated upregulated PD-1/PD-L1 axis in schistosomal infection, and we emphasise the value of IHC analysis as a cost-effective, broadly available diagnostic modality, especially in developing countries where such diseases are most prevalent. Therefore, analogous to cancer settings, immunohistochemistry can be used to predict the immune checkpoint pathway status in schistosomiasis. It is worth noting, however, that while this study sheds light on the involvement of the immune checkpoint pathway in human tissues infected by schistosomiasis, a better understanding of the pathogen’s microenvironment could be achieved through the use of expanded marker panels, including additional immune checkpoint and inflammatory cell markers, where additional efforts could be directed towards evaluating the targeted immune responses while disregarding non-specific surrounding immune infiltration.

To conclude, the discovery of the PD-1/PD-L1 pathway has revolutionised the field of medical oncology; it constituted the base of immunotherapy that has shown remarkable promise in various cancers.⁴⁹ Whether the potential benefit of immune regulatory approaches such as PD-1/PD-L1 inhibitors as well as CAR-T cell therapy could be expanded beyond the cancer scope to include inflammatory and infectious diseases including schistosomiasis or tuberculosis requires immense further investigation. Moreover, considering that PD-1/PD-L1 status and immune responses are incredibly dynamic processes where an intricate balance between antipathogen activity and prevention of autoimmunity is finely maintained by inflammatory cells and their mediators often serving the host’s advantage, significant effort must be devoted to determining which stages of infection could benefit most from therapeutic immunomodulation without undesirable aggravated pathology.

Data availability statement

Data are available upon reasonable request.