Macrophage Dysregulation in Hemophagocytotic Lymphohistiocytosis: From Cytotoxic Failure to Hyperinflammatory Cascade

Priyanjan Banerjee

doi:10.20944/preprints202610.0886.v1

Submitted:

08 October 2026

Posted:

10 October 2026

You are already at the latest version

Abstract

Hemophagocytotic lymphohistiocytosis (HLH) is a rare but often life-threatening hyperinflammatory syndrome which is characterised by pathological macrophage activation and failure of self-regulation in immunity. This review examines the key role in macrophages as both victims of defective cytotoxic control, but instead of staying as passive victims, become overactivated partners in immune-mediated tissue damage and primary executors of end-organ damage in HLH. Here, exploration of the links between the impaired perforin-granzyme cytotoxicity, sustained presentation of antigen, and the ultimate culminating into M1 macrophage polarization which drives the clinical conditions of fever, cytopenia, organomegaly, and coagulopathy has been studied. Understanding the dysfunction of macrophages in HLH provides critical insights for targeted therapeutic interventions in this life-threatening rapidly fatal syndrome. Also, the therapeutic targeting of the macrophage pathways, and the future therapeutic developments that are needed to be taken into consideration are discussed, followed by the immediate clinical relevance of macrophage-centric understanding of HLH.

Keywords:

Hemophagocytotic lymphohistiocytosis (HLH)

;

perforin

;

granzymes

;

cytotoxicity

;

macrophage

;

polarization

Subject:

Medicine and Pharmacology - Hematology

Introduction

The ability of the immune system to mount an effective response against the pathogens, and at the same time maintaining self-tolerance depends on precise regulatory mechanisms that can control and terminate inflammatory responses, after neutralizing the threats. In an individual with healthy immunity, cytotoxic lymphocytes like the CD8+ T cells and Natural Killer (NK) cells, are not just killer cells but also act as supervisors. After the successful termination of an APC (Antigen Presenting cell), this serves as a stop signal for the rest of the immune system [1]. In this process, they utilize the perforin-granzyme pathway, and the synergy between these two leads to the death of the target cells. This is crucial for understanding of the immune homeostasis.

Hemophagocytotic lymphohistiocytosis (HLH) arrives in scene after a failure in the functional regulatory system. In both familial forms (due to mutations in the genes involved in cytotoxic pathway) and secondary forms (triggered by the infections, malignancies and autoimmune diseases), the inability to properly terminate immune responses leads to pathological macrophage hyperactivation. The Macrophage Activation Syndrome (MAS) proposes a defect in the lymphocyte cytolytic activity. The cytokine storm, resulting in the activation of macrophages, causes hemophagocytosis, and ultimately culminates to multi-organ dysfunction [2].

The Perforin Problem

Normal Cytotoxic Function

The perforin–granzyme system is one of the mechanisms that have been applied by the NK cells, responsible for killing the infected cells, also one of the most interesting mechanisms of the immune system, where self regulation is achieved. Perforins are stored in the NK cells and CTLs in specialised vesicles called lytic granules, a type of secretory lysosome. These function as a pore forming protein, acting as a drill, puncturing holes in the membrane of the target cell, creating channels in target cell membranes. The pores formed allow the granzymes, which are serine proteases with a high potency in pro-apoptotic activity, to enter the target cells and trigger apoptosis or programmed cell death [3].

Under normal conditions, this causes the elimination of infected cells, tumor cells, and, most importantly, overactivated antigen-presenting cells which include macrophages. The ability of the cytotoxic lymphocytes to cause the killing of hyperactivated macrophages prevents the sustained production of cytokine that otherwise leads to pathological inflammation. These mechanisms ensure that immune responses are in perfect coordination, scaled and time-limited.

Cytotoxic Failures in HLH

In case of Familial HLH, mutations in genes encoded with key components of granule-mediated cytotoxic pathway, that includes PRF1 (perforin), UNC13D, STX11, STXBP2, and RAB27A can cause impairment in the ability of cytotoxic lymphocytes to eliminate antigen-presenting cells [4]. Due to the failure of the killing of the infected or stressed cells, and the failure to remove the macrophages by the cytotoxic ‘feedback’ control, the defect results in the creating of a situation of persistent presentation of antigen.

The results of the cytotoxic failure has proved to be far beyond the impaired pathogenic clearance. Due to the failure of the CTLs and NK cells in killing, sustained antigen presentation poses the relentless production of massive quantities of interferon-gamma (IFN-γ) and other cytokines. This results in the creation of a positive feedback loop, due to which the macrophages are further activated and left in an activated hemophagocytotic state, and amplification of pro-inflammatory cytokine cascade, defining HLH [5].

Macrophage Hyperactivation

Cytokine-Driven Activation

The activation of macrophage in HLH is driven fundamentally by the activity of the cytokines that’s dominated by IFN-γ. It is produced primarily by the CD8⁺ T-cells, and NK cells in response to persistent presence of viruses, bacteria, or tumor cells, which presents antigens. It is a key regulator in the activation of the macrophages, which literally ‘wakes up’ the macrophages, turning them into highly efficient killing machines that swallow and digest pathogens (phagocytosis).

Defective Cytotoxicity (in NK cells and CD8⁺ T cells) prolongs contact with the antigen-presenting cells, causing the excess production of IFN-γ and others including tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-18. These mediators work in synergy to maintain macrophage activation, while promoting the release of additional inflammatory factors. Defective killing of the target cells and persistent immune triggers keep T cells and macrophages activated, creating a vicious positive loop of cytokine production and inflammation [6].

M1 Polarisation and Pathological Consequences

In recent studies, it has been noted that during the introduction of lipopolysaccharide (LPS) in a mouse model with HLH-like characteristics, a prolonged increase of M1 (classically activated) macrophage proportion, while there is a delay in the increase of M2 (alternatively activated) macrophage proportion. This imbalance in the polarization gives rise to consequences like severe anaemia, thrombocytopenia and conditions like impaired hematopoiesis and systemic inflammation. HLH/MAS is driven by Th1 cytokines (IFN-γ, TNF-α, IL-2) and uncontrolled macrophage activation, consistent with an M1-skewed environment [7].

M1-polarized macrophages in HLH exhibit increase in the production of pro-inflammatory mediators, and results in increase of hemophagocytotic activity, which is the process which involves the pathological consumption of the blood cells. M1 macrophages cause release of cytokines that causes inhibition of the proliferation in the surrounding cells, damaging contiguous tissues, while M2 macrophages release cytokines which do the reverse, i.e. promote proliferation of contiguous cells and repairing of tissues. M1-M2 pathway, being a tightly controlled process, an imbalance of macrophage M1-M2 polarization and failure to transiting to anti-inflammatory responses prevents the resolution of inflammation and restoring of normalcy in homoeostasis in hematopoietic microenvironment is not possible.

Monocyte Hypersensitivity

In case of patients with secondary HLH/MAS, peripheral monocytes show increased basal and IFN-γ stimulated STAT1 phosphorylation, which appears as increased inflammatory subsets and exaggerated cytokine responses to microbial or endogenous stimuli. This contributes to the severity and the persistence of the hyper inflammatory state [8].

Clinical Manifestation of Macrophage Dysregulation

Here are the features of the HLH that can directly be traced to macrophage Hyperactivation, summarised in Table 1:

Cardinal Clinical features and Macrophage Mechanisms

Table 1. Clinical features of HLH and their macrophage-mediated mechanisms [2].

Clinical feature	Macrophage-mediated Mechanism
Fever	Overproduction of TNF-α, IL-1, IL-6, IL-18, IFN-γ in the cytokine storm; activated macrophages and T cells produce these pyrogenic cytokines and drive sustained high fevers
Cytopenias (pancytopenia)	Bone marrow and tissue hemophagocytosis by activated macrophages; excessive cytokines (e.g., IFN-γ, TNF-α) also suppress hematopoiesis and contribute to consumptive anemia and pancytopenia
Splenomegaly / hepatosplenomegaly	Expansion and infiltration of hemophagocytic macrophages and lymphocytes in spleen and liver, with histiocytic hyperplasia and lymphohistiocytic infiltration
Hyperferritinemia	Massive ferritin production by activated macrophages (CD68⁺/IL-12⁺/CD163^low early on) under stimulation by TNF-α, IL-1β, IL-6, IL-18 and NF-κB-dependent pathways; ferritin may further amplify IL-1β and cytokine storm
Hypofibrinogenemia	Activated macrophages increase plasminogen activators, promoting fibrinolysis and hypofibrinogenemia; cytokine storm and liver dysfunction contribute to DIC-like coagulopathy
Elevated sCD163 (and sCD25)	Intense activation of CD163⁺ macrophages leads to shedding of CD163 into plasma (sCD163); high levels track macrophage activation and correlate with hyperferritinemia and high IL-6/IL-18/IFN-γ in sepsis-associated MAS and hyperferritinemic sepsis

Biomarkers of Macrophage Activation:

Ferritin has the most important function as the main iron storage protein, but studies have showed that it can act as an immune and inflammatory mediator. Very high ferritin, with levels exceeding 10,000 ng/mL, are highly specific features for HLH, reflecting a profound degree of macrophage activation, and is a key feature of several life-threatening cytokine-storm conditions [9].

In other cases, elevated soluble CD163 (sCD163) represents a 130 kDa scavenger receptor which are expressed almost exclusively on monocytes, particularly tissue macrophages. Elevated sCD163 appears across many inflammatory, metabolic, infectious conditions involving liver and malignancy, correlating to severity in diseases, fibrosis and disease prognosis [10]. These biomarkers are also involved in shaping the macrophage function and is an emerging therapeutic target for both inflammatory diseases, including cancer. Other additional emerging biomarkers include IL-18 and CXCL9, reflecting different aspects of inflammatory cascade and the pathway activity of IFN-γ [11].

Therapeutic targeting of Macrophage Pathways

Current Therapeutic Approaches

Macrophage is highly recognized as a central effector for HLH, which has guided the development in the targeted therapeutic strategies. The HLH-94 and HLH-2004 protocols, forming the backbone of the current treatment, target the activation of the macrophages through multiple mechanisms:

Dexamethasone: Involved in the suppression of macrophage activation and cytokine production by selectively stimulating glucocorticoid receptors, altering gene transcription to reduce pro-inflammatory cytokines (e.g., TNF, IL-1, IL-6), and increasing anti-inflammatory factors [12].
Etoposide: It helps in depleting both activated lymphocytes and macrophages by stabilizing the transient topo II- DNA complex, leading to single and double DNA breaks and cell cycle arrest in the S/G2 phase, that triggers apoptosis or programmed cell death [13].
Cyclosporine A: It has the property to work upstream by inhibiting the activation of T cells, and suppresses T cell response through cyclophilin A. Thus it reduces the cytokine signals that drive the hyperactivation of macrophages [14].

This is a prolonged approach that reflects the complex, interconnected nature of the pathological inflammatory network in HLH.

Emerging Targeted Therapies

There has been recent advances in the understanding of the HLH pathophysiology which has led to the development of more specific intervention which target key cytokine pathways, summarised in Table 2.

Therapeutic Gaps

Despite recent advances in the field, there are significant therapeutic gaps. While the macrophage activation can be suppressed by the systems, strategies to actively repolarize M1 macrophages towards anti-inflammatory M2 phenotypes have not yet been clinically validated. This represents an area for a future development, as promotion of resolution of inflammation can be as important as the initial suppressing of it.

Experimental Evidence for Macrophage Centric Pathogenesis

Mouse Models of HLH

Experimental studies in Anti-CD40 models, agonistic anti-CD40 antibody selectively activates LysM⁺ macrophages and causes induction of an entire HLH/MAS-like syndrome (cytokine storm, hyperferritinemia, high sCD25, erythrophagocytosis, endothelial activation, cytopenias), that too on an otherwise normal background [18].

Transgenic Models

A HIF1A-inducible transgenic line that expresses HIF1A/ARNT in case of hematopoietic cells develop lethal HLH-like diseases (including anaemia, thrombocytopenia, splenomegaly, multi-organ failure) on induction, with type 1-polarisation in macrophages and NK dysregulation. This phenotype is not dependent on adaptive immunity and IFN-γ, which places HIF1A as a downstream, innate mediator in case of HLH [19].

Future Directions and Conclusions

The central role of macrophage dysregulation in HLH provides an unifying framework for the understanding of the complex syndrome. From the initial cases of cytotoxic control failure, to the sustained hyperinflammatory cascade, and ultimately leading to clinical manifestations, macrophages serve as both targets on one hand, and mediators of pathology on the other.

Research Priorities

Future therapeutic developments should focus on:

Clarification of macrophage-centric pathways: More defining of macrophage-intrinsic vs T-cell–driven mechanisms, including how and when macrophages can act as independent drivers rather than passive responders [20].
Understanding the dynamic M1/M2 and intermediate phenotypes, which include hemophagocytic macrophages evolve over the course of the disease.
Biomarkers of macrophage dysregulation, validating of soluble CD163, ferritin, free IL-18, Cytokine panels and flow markers distinguish HLH/MAS from conditions like sepsis and other hyperinflammatory states [21].
Further genetic dissection of MAS/secondary HLH (including inflammation genes, viral control and metabolism) for explaining macrophage dysregulation threshold.

Clinical Relevance

The macrophage centric understanding of HLH has some immediate clinical relevance:

Recognizing HLH/MAS as a macrophage-driven cytokine storm supports the application of ferritin, soluble CD25, and macrophage-driven markers (sCD163), as these levels reflect a macrophage activation state.
Understanding that therapeutic efficacy, or macrophage-focused strategy includes IL-1 blockade (anakinra), IL-6 blockade and IFN-γ neutralisation (emapalumab) in sHLH/MAS and cytokine storm settings.
Understanding that application of glucocorticoids in modulating the signalling of macrophages, affecting both the biomarker interpretation and the activity of the diseases.

Conclusions

It can be safely stated that HLH is a disease that results from the failure of one of the most fundamental mechanisms of the immune system, the ability to terminate inflammatory responses in an appropriate manner. By targeting the particular consequences of the failure propagated by macrophages, current and future therapeutic techniques can be developed, which offer improved outcomes for this rapidly fatal but potentially treatable syndrome. This evolution in the understanding of the biology of the macrophages shows the importance of basic immunological insights in translating into therapeutic advances. With this gradual unravelling of the complex networks that govern macrophage activation and resolution, more effective, and targeted interventions for this challenging disorder can be achieved.

Author Contributions

The author, Priyanjan Banerjee conceived the topic, performed the literature search, drafted the initial manuscript and revised the final text.

Funding

The author declares that no grants, funds, or other support were received during the preparation of this manuscript.

Data Availability Statement

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Conflicts of Interest

The author has no relevant financial or non-financial interests to disclose.

Ethical approval and consent to participate

Not applicable.

Use of Large Language Model (LLMs)/Artificial Intelligence (AI)

The author hereby confirms that no generative AI tools or Large Language Models (LLMs) were used for content generation in the creation of this manuscript.

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Table 2. List of therapies, target and interventions, targeting key cytokine pathway.

Therapies	Target	Explanation
Emapalumab	Direct IFN-γ blockade	A fully human monoclonal antibody that fully binds and neutralizes IFN-γ, the key macrophage –activating cytokine in MAS/HLH, blocking the signal, showing efficacy in refractory cases [15]
Ruxolitinib	JAK1/2 pathway inhibition	An oral JAK1/2 inhibitor that blocks intracellular signalling downstream of IL-2, IL-6, IL-10, IFN-γ [16]
Anakinra	IL-1 receptor antagonism	A recombinant IL-1 receptor antagonist that competes with IL-1α/IL-1β for IL-1R1, preventing downstream NF-κB activation and secondary cytokine/chemokine production [17]

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