Heat-Killed Lactic Acid Bacteria Enhances Human NK Cell Cytotoxicity in Chronic Myeloid Leukemia Cells through IL-32α-Mediated Cell Death

Abstract

Abstract Heat-killed lactic acid bacteria (K-LAB) are known to be important immunomodulators that stimulate tumor necrosis factor-α (TNF-α) and nitric oxide (NO) production as well as increase phagocytic activity in macrophages. Natural killer (NK) cells play a critical role in innate immune response and induce spontaneous killing of tumor cells and virus-infected cells. However, the effect of K-LAB on NK cells is still unclear. In this study, we investigated the effect of K-LAB on blood lymphocyte (PBL) cytolytic activity because only NK cells are able to kill tumor cells and virus-infected cells spontaneously among the PBLs. We found that K-LAB stimulated PBL cytolytic activity in dose- and time-dependent manners. Especially K-LAB stimulates IL-32α expression in PBLs. Interleukin-32 (IL-32) was recently identified as a pro-inflammatory cytokine that is induced by IL-18 in natural killer (NK) cells and IL-32 is known as the most abundant transcript. To test the effect of IL-32 in the immune regulation, we constructed IL-32α-overexpressing chronic myeloid leukemia (CML) cell lines, Kcl22 and BV173. Interestingly, IL-32α-overexpressing Kcl22 and BV173 showed higher NK cell-mediated killing. Flow cytometry analysis revealed that overexpression of IL-32 induced increased expression of Fas and UL16-binding protein 2 (ULBP2) in CML cells. In addition, the transcription factor Ets-1 plays a key role in ULBP2 specific expression by IL-32α overexpression in ULBP family members. These data means that IL-32 α stimulates Fas and ULBP2 expression via activation of Ets-1, which increases NK susceptibility of CML cells. Taken together, K-LAB stimulates NK cytolytic activity through stimulation of IL-32α production, which enhanced NK cell susceptibility of CML cells.

Killed lactic acid bacteria (사균체; K-LAB) tumor necrosis factor-α (TNF-α)와 Nitric oxide (NO) 생성을 자극하는 중요한 면역조절물질로 macrophage에 포식활성을 증가키는 것으로 알려져있다. Natural killer (NK) cell은 선천성면역반응에 중요한 역할을 수행하고 종양세포와 바이러스에 감염된 세포의 자연살상을 유도한다. 그러나 NK cell에 사균체의 효능은 아직 밝혀지지 않았다. 본 연구에서는 오직 NK cell만이 blood lymphocyte(PBL) PBLs의 사이에서 tumor cell과 virus-infected cells을 자발적으로 죽일 수 있기 때문에 PBL cytolytic activity에 대한 사균체의 효능을 조사하였다. 그 결과 사균체가 용량과 시간에 의존적으로 PBL cytolytic activity을 자극한다는 것을 알았고, 특히 사균체는 PBLs에 IL-32α 발현을 자극한다. Interleukin-32(IL-32)는 최근 NK cell에서 IL-18에 의해 증가되는 proinflammatory cytokine으로 확인되어졌고, IL-32는 가장 풍부한 transcript로 알려졌다. 면역조절에 대한 IL-32α의 효능을 실험하기 위해, IL-32α가 과발현된 chronic myeloid leukemia(CML) cell lines인 Kc122와 BV173을 설계했고, 흥미롭게도 IL-32α가 과발현된 CML cell line인 kc122 와 BV173이 더 높은 NK cell-mediated killing을 보였다. Flow cytometry analysis은 IL-32α의 overexpression 이 CML cell내 Fas와 UL16-binding protein2(ULBP2)의 발현 증가가 야기된 것을 나타내었다. 게다가 transcription factor Ets-1은 ULBP family members내 IL-32α의 overexpression으로 ULBP2의 제한적 발현에 중요한 역할을 수행한다. 이들 데이터는 IL-32α가 Ets-1의 활성화를 통하여 fas와 ULBP2 발현을 자극하고, 그로인해 CML cell의 NK susceptibility가 증가한다는 것을 의미한다. 결과적으로, 사균체는 IL-32α production의 자극을 통하여 NK cytolytic activity을 자극하고, CML cells의 NK cell susceptibility를 높힌다.