The innate immune system functions as the body’s first line of defence through the initiation of a nonspecific rapid response to pathogens and infections, while also serving a critical role in the overall regulation of tissue homeostasis and immunity surveillance (7). Monocytes, a subset of white blood cells originating from hematopoietic stem cells (HSCs) in the bone marrow, patrol the bloodstream for sites of inflammation and, upon migration to tissues, differentiate into their tissue-specific macrophages (9). Once relocated into the tissue, these differentiated and highly plastic macrophages undergo polarization in adaptation to their specific microenvironment, a process tightly regulated under the KLF4 transcriptional control (8, 10).

In principle, there are two subsets of macrophages, namely tissue resident and non-resident macrophages. Resident macrophages (Ly6C^lo/CCR2^-), originating from yolk sac-derived erythro-myeloid progenitors, predominantly exhibit an M2-like anti-inflammatory phenotype under homeostatic conditions, contributing antagonistic roles of inflammation, such as mitigating insulin resistance, eliminating parasites, and promoting tissue remodelling and repair (11–13). In contrast, non-resident (blood-borne infiltrating) macrophages, derived from circulating (Ly6C^hi) monocytes recruited by the CCL2-CCR2 chemotaxis pathway, are more likely to adopt an M1-like proinflammatory phenotype to enhance pathogen clearance and increase blood vessel permeability and the activation of other inflammatory mediators (14–17). Recent studies have established the crucial role of KLF4 in modulating macrophage polarization, influencing the balance between proinflammatory M1 and anti-inflammatory M2 phenotypes, thus contributing to immune homeostasis and the resolution of inflammation (1).

Monocyte differentiation is embedded within the broader framework of haematopoiesis – a highly transcriptionally regulated process that drives the specialization of HSCs into various blood lineages. HSCs first give rise to common myeloid progenitors (CMPs), which differentiate into granulocyte-macrophage progenitors (GMPs). GMPs can then commit to either granulocytic or monocytic lineages depending on specific transcription factor cues (18).

The myeloid differentiation process is modulated by the induction of lineage-restricted transcription factors such as PU.1, a member of the ETS family of transcription factors, that bind to consensus GGAA-like motifs, for GMPs differentiation and GATA-1 as an antagonist to drive megakaryocyte-erythroid progenitors (MEPs) differentiation (19–21). KLF4, a downstream target gene of PU.1, binds the CD14 promoter, thus specifying monocyte commitment. In PU.1-deficient mouse fetal liver cells, KLF4 overexpression restores monocyte differentiation in the absence of PU.1, supporting a partially compensatory or synergistic relationship between the two transcription factors (22) ( Figure 2 ). Thus, KLF4 acts as both a determinant and effector of monocyte lineage commitment and differentiation. The expression of KLF4 is present in monocyte-lineage cell lines (e.g., THP-1, U-937) but absent in other hematopoietic cell types, underscoring its specificity.

Functionally, KLF4 not only induces monocytic surface markers (CD11b, CD14; while not impacting granulocytic and lymphocytic markers) but also causes morphological changes (23). These characteristic changes in cellular morphology, include increased cytoplasmic size, smaller and more condensed nuclei, and ruffled cell edges, consistent with the acquisition of a monocytic phenotype (7, 24). Understanding these regulatory mechanisms not only advances our knowledge of hematopoietic lineage commitment but also provides potential targets for therapeutic modulation in inflammatory and myeloid-related disorders.

Macrophages can undergo polarization in adaption to their specific microenvironment, a process tightly regulated by KLF4 (10). The heterogeneous cell population of macrophages plays an essential role in the innate immune response due to their remarkable plasticity, allowing them to adopt distinctive functional phenotypes (M1/M2) in response to tissue environment cues governing polarization (25, 26). This binary classification of macrophages is an oversimplification of a more dynamic spectrum of activation states. Given its regulatory role, KLF4 serves as a key transcription factor essential in modulating and regulating macrophage polarization, to facilitate tissue repair but also suppresses excessive inflammation, highlighting its dual role in immune homeostasis (27).

Neutrophil granules are membrane-bound vesicles storing pro- and anti-inflammatory molecules, that are released through a process termed degranulation to execute pathogen elimination and tissue remodelling functions (43). Among these molecules are essential antimicrobial and cytotoxic molecules, such as matrix metalloproteinase-9 (MMP-9) and myeloperoxidase (MPO) (44). In wild-type neutrophils, lipopolysaccharide (LPS) stimulation upregulates MMP-9 mRNA, promoting granule release. However, KLF4-deficient neutrophils exhibit a significant reduction in MMP-9 mRNA levels, indicating that KLF4 is necessary for proper transcriptional regulation of granule-associated proteins. Furthermore, reduced secretion of MMP-9 and MPO in these neutrophils suggests that KLF4 deficiency primarily disrupts granule content; since MMP-9 mRNA levels were already suppressed in KLF4-deficient neutrophils, this deficiency in secretion is not due to impaired degranulation but rather to a reduction or defect in granule content (45).

Beyond granule-associated proteins, KLF4-deficient neutrophils also exhibit altered cytokine secretion. Proinflammatory cytokines such as TNF-α, keratinocyte chemoattractant (KC), and IL-1β, along with the anti-inflammatory cytokine IL-10, coordinate the innate immune response (46). Upon Streptococcus pneumoniae stimulation, KLF-deficient neutrophils showed significantly reduced TNF-α and KC secretion, while IL-10 release was elevated compared to the wild-type control. IL-1β levels remained unaffected, possibly suggesting that KLF4 selectively regulated specific cytokine pathways in neutrophils. Notably, these changes were observed in blood-derived PMNs but not in remaining white blood cells (WBCΔPMNs), indicating potential differences in KLF4-dependent cytokine regulation amongst neutrophil populations (47). Together, these findings highlight KLF4’s essential role in promoting proinflammatory cytokine release while restraining anti-inflammatory signalling, making it a critical regulator of neutrophil-mediated immune responses.

Effective bacterial clearance mechanisms are essential for neutrophils to maintain host defence from bacterial infections (48). The loss of KLF4 disrupts these processes, compromising immune responses and increasing susceptibility to infection. Following intraperitoneal E. coli infection, myeloid-specific KLF4-deficient mice exhibited significantly higher mortality rates than controls. Increased bacterial burden in these mice points to uncontrolled infection and subsequent development of bacteraemia. Furthermore, circulating levels of TNF-α, MPO, and MMP-9 were significantly lower in myeloid-specific KLF4-deficient mice, signifying a diminished host defence mechanism. Impaired bacterial killing capabilities in KLF4-deficient neutrophils ex vivo further support this observation (45). KLF4-deficient murine polymorphonuclear neutrophils (PMNs) also exhibit reduced pneumococcal killing. After incubation with opsonized S. pneumoniae D39 or R6× for three hours, KLF4-deficient blood-derived PMNs showed significantly reduced bacterial clearance (47). Although the precise mechanism by which KLF4 regulates neutrophil antimicrobial function remains unclear, these findings suggest that KLF4 plays a central role in coordinating the transcriptional response required for effective bacterial killing (49).

Neutrophil function is protective in bacterial killing, but excessive systemic inflammation can lead to septic shock and death (50). Interestingly, while KLF4 deficiency impairs the immune response to bacterial infection, it appears to confer resistance to excessive inflammation in response to direct challenge with endotoxin (45). LPS-induced mortality rate is greatly reduced in myeloid-specific KLF4-deficient mice compared with the control, along with lower levels of pro-inflammatory factors MPO and TNF-α. Beyond acute infection, neutrophils also play a key role in chronic inflammatory diseases, where their interactions with other immune cells influence disease progression (51). Specifically, neutrophils may influence chronic inflammation in the experimental autoimmune encephalomyelitis (EAE) model, which mimics multiple sclerosis (MS) in humans (52, 53). KLF4-deficient neutrophils impaired chronic inflammation, exhibited delayed disease onset, and significantly reduced EAE severity compared to wild-type controls despite a 100% incidence. At the disease onset phase, a significant reduction in CNS-infiltrating neutrophils, T helper cells, B cells, and dendritic cells was observed in myeloid-specific KLF4-deficient mice, further suggesting that KLF4-deficient neutrophils are defective in mediating chronic inflammation (45). These findings reinforce the role of KLF4 in promoting inflammatory responses, with its absence reducing acute endotoxin-induced inflammation and chronic autoimmune-mediated neuroinflammation.

Previously, TLR4 activation by LPS has been shown to induce KLF4 expression in murine neutrophils (45). KLF4-deficient neutrophils exhibited impaired responses to LPS stimulation, which typically activates TLR4 and initiates a signalling cascade leading to the activation of the IкB kinase (IKK) complex. This complex phosphorylates IкBα, resulting in its degradation and the subsequent release of NF-кB, a key transcription factor of pro-inflammatory genes (54). In KLF4-deficient neutrophils, LPS-induced phosphorylation and degradation of IкBa are significantly attenuated, resulting in reduced NF-кB activation and decreased transcription of its target genes such as TNF-α (45). One of the key mechanisms by which KLF4 influences this pathway is through the regulation of CD14, a crucial co-receptor for TLR4 (55). CD14 aids TLR4 in recognition of bacterial components like LPS, and typically, neutrophils upregulate CD14 levels upon encountering LPS. However, in KLF4-deficient neutrophils, both CD14 mRNA and surface protein expression were reduced, both at baseline and after LPS activation. This reduction in CD14 expression impairs the ability of neutrophils to efficiently recognize bacteria stimuli, weakening the activation of the downstream TLR4-NF-кB signalling cascade. While other components of the TLR4 pathway remain unaffected by KLF4 deficiency, the decreased expression of CD14 is a critical factor in the reduced responsiveness to LPS in neutrophils (45) ( Figure 3 ).

Other findings testing various TLR pathways (TLR2, TLR4, and TLR9) demonstrated that activation of a single TLR by its respective agonist (MALP-2, LPS, or CpG) was insufficient to induce KLF4 expression in neutrophils, macrophages, or epithelial cells (47). Possible explanations for the difference in findings is that mouse and human neutrophils respond to LPS differently from variations in TLR4 or the use of different types of LPS from different bacterial sources. Additionally, neutrophils do not always respond strongly to a single stimulus but can become more reactive after priming (56). This suggests that multiple stimuli might be needed to induce KLF4 expression, which was observed with S. pneumoniae stimulation (47).

Functional CD8+ T cells, otherwise known as cytotoxic T cells or killer T cells, originate from naïve CD8+ T cells in the bone marrow and undergo a rapid expansion, differentiation, and selection in the thymus (61). Once matured, cytotoxic T cells become mono-specific CD8+ T cells capable of contributing to the immune response. Novel research indicates following enhanced T cell receptor (TCR) activation by TCR crosslink, naïve CD8+ T cells in Klf4-deficient mice demonstrated increased proliferation (60). In the same experiment, these Klf4-deficient naïve CD8+ T cells also increased in proliferation in response to bacterial infection – specifically, when upon infection with a strain of Listeria monocytogenes-OVA, Klf4-deficient naïve CD8+ T cells generated more memory CD8+ T cells in both the primary and secondary responses to the infection. Additionally, circulating CD8+ T cells remain quiescent when under KLF4 expression (62) maintained through activation of p21, a kinase inhibitor involved in cell cycle regulation (63). CD8+ T cells from Klf4-deficient mice upregulated proliferation following in vitro TCR crosslink activation and increased in vivo homeostatic population expansion in the spleen. The presence of KLF4 modulates repression of T cell development in T ALL by inducing apoptosis through suppression of the BCL2/BCLXL genes ( Figure 4 ). Previous studies also reveal that the KLF4 gene becomes hypermethylated in cases of T ALL, while forced KLF4 overexpression suppresses its downstream signals, inhibiting T ALL progression and inducing T cell apoptosis (64). Mechanistically, through the binding of KLF4 to the promoters of T cell-associated genes, NOTCH1, BCL2, and CXCR4, repression and inhibition of T Cell proliferation is initiated (65). The results of these in vitro experiments posit KLF4 as a negative regulator in T cell-associated genes and, therefore, T cell proliferation. Specifically, KLF4 functions as a negative regulator in the proliferation of (naïve) CD8+ T cells under increased TCR activation/stimulation in vitro. Disruption of KLF4 activity enhances proliferation, and KLF4 also plays a key regulating role in the proliferation of functional memory CD8+ T cells.

B Cells exist naturally in the quiescent state, non-dividing and inactive until they encounter the appropriate antigen. In active B cells, KLF4 becomes heavily downregulated, and in vitro studies indicate that an induced KLF4 expression prevents and decreases B cell cycle progression to the S phase via the following modulation of the known target genes of KLF4: increased p21 activity and decreased cyclin D2 and c-Myc activity (66). Forced expression of KLF4 also increased cell death in those proliferating B cells. However, the KLF4-deficient B cells displayed similar survival rates and expression as normal B cells, suggesting that the loss of KLF4 had no noticeable impact on B cell proliferation and development. It appears KLF4 serves a redundant role as other members of the KLF family, like KLF2/KLF3, which are also highly expressed in naïve B cells and downregulated upon activation, and loss of KLF4 can be compensated by other KLF members. KLF4’s function is reminiscent of different members of the FOXO protein family, like FOXO1 and FOXO3a, which induce cell cycle arrest to G1 and increase apoptosis upon forced expression (67). The FOXO protein family may be involved in KLF4 transcription, and KLF4’s regulation by FOXO proteins is a critical mechanism that determines B cell proliferation and survival.

In another study, KLF4 is shown to inhibit B cell proliferation in patients with B cell lymphomas, like Burkitt Lymphoma, follicular lymphoma, and classic Hodgkin lymphoma (68). Primary cases of B-cell lymphomas revealed that the KLF4 promoter was methylated, thus silencing the expression of KLF4 in the B cells ( Figure 4 ). By overexpressing KLF4 in Burkitt lymphoma cell lines, researchers were able to induce cell cycle arrest in G_o and G₁, pausing the proliferation of B cells. In Hodgkin’s lymphoma cell lines, KLF4 overexpression resulted in increased apoptosis through the activation of the BAK1 proapoptotic gene. BAK1 activity is sequestered by the MCL1 and BCL2L1/BCL_-XL pathways, and is only functional if it exists in higher concentrations than the sequestering capabilities of its regulators. KLF4 may be able to overstimulate BAK1 production, forcing it to become active and induce apoptosis in B cells. Interestingly, KLF4 also has a strong regulatory effect on the MSC/ABF-1 repressor, which is highly expressed in classic Hodgkin’s Lymphoma, follicular lymphoma, and Burkitt Lymphoma (69, 70). MSC/ABF-1 is a helix-loop-helix protein that suppresses the transactivating potential of E-box transcription factors, E47/E12 (71) which are downstream proteins involved in B cell proliferation. E47/E12 regulate proliferation by activating the p21 gene (72). It is plausible that KLF4 controls the proliferation of B cells by inhibiting the MSC/ABF-1 factor, which increases activity of E47/E12, which in turn activates the p21 protein and delays the cell cycle.

During infection, DCs are responsible for initiating the adaptive immune response, activating T cells by capturing and presenting target antigens (73, 74). There are several subsets of DCs, like conventional DCs (cDCs), Langerhans cells (LCs), monocyte-derived DCs, among others, all having specific functions in response to infection/disease (75, 76) ( Figure 4 ).

In DC differentiation, KLF4 expression positively correlates with the proinflammatory characteristics of that DC subset. KLF4 activity is induced in dermal DC and monocyte-derived DC differentiation, but is repressed to enable differentiation in LCs (77). Specifically, epithelial Notch signalling represses KLF4 in developing LCs, causing Runt-related transcription factor 3 derepression in response to TGF-β1, which ultimately enables differentiation into LCs due to low cytokine expression markers. KLF4 is also important in cDC development and function, particularly in IRF4-expressing cDCs that promote Th2 immune responses. In vivo cDCs with conditional deletion of KLF4 demonstrated impaired Th2 cell response to infection by Schistosoma mansoni and house dust mites, although Th1/Th17 cell responses were unaffected in response to other infections (78). In different tissues, KLF4 deletion decreased expression of IRF4 in pre-cDC subsets, and caused selective loss of cDC subsets expressing IRF4.

KLF4 activity also modulates inflammatory immune responses, including the production of inflammatory molecules like IL-6 by DCs. Rosenzweig et al. found that KLF4 plays dual functions to modulate expression of IL-6, directly activating the IL-6 promoter and remodelling chromatin (79). Further, they showed that DCs lacking KLF4 had significantly reduced levels of IL-6 mRNA and protein, although IL-6 was not fully absent.

Natural Killer (NK) cells are fundamental cytokines in the innate immune response pathway to cancer (79), inhibiting proliferation and migration/colonization of distant tissues to combat primary tumour cells and metastasis (79). NK cells also produce large amounts of cytokines like interferon-γ, modulating adaptive immune responses and participating in similar pathways (80, 81).

KLF4 has been shown to promote survival of NK cells in the spleen, and also maintain the number of cDCs in the spleen (82) mutated the KLF4 gene in cre-transgenic mice, and found that somatic deletion leads to heavily reduced numbers of NK cells in the blood and spleen but not in bone marrow (BM), liver, or lymph nodes (82). Functional and immunophenotypic analyses suggested increased NK cell apoptosis in these cells, and that survival is dependent on BM-derived hematopoietic cells from the spleen. Further, numbers of CD11c^hi DCs, which promote NK cell survival, were significantly reduced in the KLF4-deficient mice, suggesting that the KLF4 gene is associated with the maintenance of spleen DCs, which support differentiation and survival of NK cells.

KLF4 additionally plays a role in upregulating NK cell ligands, specifically the NKG2D ligand MICA, which stimulates immune responses of NK cells ( Figure 4 ). Alkhayer et al. determined that the MICA promoter contains KLF4-binding motifs, and that in acute myeloid leukaemia (AML), KLF4 mediates inducible expression of MICA (83). Upon inhibition/ablation of KLF4, HDAC-mediated upregulation of MICA is also reduced. Moreover, the APTO253 molecule, which is a known KLF4 inducer, was found to upregulate MICA in AML cells. APTO253-treated AML cells were also rendered more susceptible to termination by NK cells.